James E. Masse1, Frederic H.-T. Allain1, Yi-Meng Yen3, Reid C.
Johnson2,3 and Juli Feigon1,2
1Department of Chemistry and Biochemistry,
2Molecular Biology Institute,
3Department of Biological Chemistry,
University of California,
Los Angeles, CA 90095-1569
The use of uniformly 13C,15N labeled RNA has greatly facilitated structural studies of RNA oligonucleotides by NMR. Application of similar methodologies for the study of DNA has been limited, primarily due to the lack of adequate methods for sample preparation. Methods for both chemical and enzymatic synthesis of DNA oligonucleotides uniformly labeled with 13C and/or 15N have been published, but have not yet been widely used. We have developed a modified procedure for preparing uniformly labeled DNA based on enzymatic synthesis using Taq DNA polymerase, that results in quantitative polymerization of the template and approximately 80% incorporation of the labeled dNTPs1. We have also developed procedures for avoiding non-templated addition of nucleotides or for their removal.
These procedures have been used to synthesize several DNA oligonucleotides, including two complementary 15 base strands, which together form a binding site for the yeast protein NHP6A. We have used heteronuclear NMR techniques, with both labeled protein and DNA, to study NHP6A and its interaction with DNA2.
References and Footnotes
1. Masse, J.E., Bortmann, P., Dieckmann, T. and Feigon,
J., Nucl. Acids Res. 26, 2618-2624, 1998.
2. Masse, J.E., Allain, F.H.-T., Yen, Y.-M., Johnson, R.C. and Feigon, J.,
J. Amer. Chem. Soc. in press.
Marion P. Olivieri1, Robert M. Wollman2 and Michael F. Swartz1
1D'Youville College,
320 Porter Avenue,
Buffalo, New York 14201
2Roswell Park Cancer Institute,
Elm & Carlton Streets,
Buffalo, New York 14263
*Author to whom correspondence should be addressed. Phone: (716) 881-7647;
Fax: (716) 881-7760; E.mail: mpo@koolnet.org and
The complete characterization of adhesion at the atomic level is of interest to the scientific community as a whole. Applications of these phenomena include the control of biofilm formation as well as cellular and tissue adhesion. Although the data related to the application of several of theses bioadhesives are available, the biophysical characterization of the adhesive molecules are seldom published.
Several proteinaceous bioadhesives, derived either from the oral cavity, the circulatory system or from marine animal exudates are known to be lysine rich. For example, poly-L-lysine is a commonly used cellular attachment agent. In addition, the blue sea mussel's exudate, mussel adhesive protein (MAP), is generally composed of a repeating decameric sequence (A-K-P-S-Y-HYP-HYP-T-DOPA-K;1). MAP contains approximately 20% lysine and is marketed as a cellular attachment agent for a variety of cell types to a various of surfaces (2).
Several structural analyses have been performed on peptides derived from MAP (e.g., 2,3,4). From studies of films formed form MAP, it has been suggested that the L-DOPA residues are responsible for protein-surface attachment, and the lysines provide the protein-cellular attachment sites (3). Along these lines, peptides were designed and characterized to better examine the structural and functional domains potentially involved in surface as well as cellular attachment.
For this study, the peptides were designed as cyclic, to limit the number of solution-state conformations the molecules could adopt. Decapeptides were synthesized that contain L-DOPA for the adhesion process. In addition, control peptides were created that contained tyrosine in place of the L-DOPA.
Surface chemical analyses were performed on films created from the peptides to evaluate their ability to attach to surfaces. The importance of the L-DOPA vs. tyrosine residues under varying pH conditions was evaluated for film formation. Ellipsometry, MAIR-IR spectroscopy and contact angle analyses data provided evidence that rinse-resistant film formation was dependent on the presence of L-DOPA at a pH 8.1 (5).
Cell attachment assays are currently assessing the ability of the peptide films to attach cells. Hanging drop crystallization experiments using protein crystal screening test solutions have identified conditions that have provided reproducible small crystals. The solutions are being refined to narrow down the optimal conditions for x-ray diffraction suitable crystals.
Two cyclic peptides {via. disulfide bonds} from SynPep (Dublin, CA) were obtained after purification and characterization by high pressure liquid chromatography and mass spectrometry (DOPA-G-C-G-G-K-A-K-G-C and Y-G-C-G-G-K-A-K-G-C). Nuclear magnetic resonance (NMR) spectra were collected for the pair of peptides. 1D and 2D COSY/TOCSY/ROESY experiments were collected on an AMX-600 MHz Brüker instrument at 295K with the L-DOPA peptide at a concentration of 5.4 mM in 90%H20/10%D2O at pH 2.74. The tyrosine-containing peptide samples were 5.4 mM at pH 3.18 in D2O and 5.4 mM at pH 2.78 90%H2O/10%D2O. The 2D experiments were collected with a data matrix of 4096 x 512 complex data points in the TPPI mode. The mixing time for TOCSY experiments was typically 50 ms while for the ROESY experiments a mixing time of 300 ms was used. The nOe data was utilized in molecular modeling software (Sybyl, Tripos, St. Louis, MO) to develop structures of these peptides within the constraints.
Chemical shift assignments of both cyclic peptides were made using a combination of NMR spectra. As evidenced by only minor differences in chemical shifts values in the N-H and C__H regions for like residues (±.01 ppm), the substitution of tyrosine for L-DOPA has little effect on the rest of the cyclic peptide. This suggests that the conformation of the cyclic portion is conserved regardless of the modified exocyclic residue. Molecular models based on the NMR data will be presented for each peptide to elucidate the effects that the modified residue has on the structure as well as it's ability to bind to surfaces under the conditions identified from the surface chemistry data.
This work was supported by a NSF MCB-9513390 grant and the NIH grant CA-16056 which supports in part the NMR Facility at Roswell Park Cancer Institute (RPCI). The authors thank Martin Lee, Jason Brice, Christopher Schafer and Davein Humphrey for their technical support.
References and Footnotes
1. JH Waite, TJ Housley and ML Tanzer, Biochem. 24:5010-5014,
1985.
2. MP Olivieri, RE Baier and RE Loomis, Biomat. (14)13:1000-1008,
1992.
3. MP Olivieri, RM Wollman and JL Alderfer, J Peptide Research, 50:436-442,
1997.
4. MK Lee, RM Wollman, SC Ceraulo and MP Olivieri (1998), "Molecular
Modeling of a 26 Residue Mussel Adhesive Protein Segment Constructed from
Overlapping Peptide Structures", The 24th Annual Meeting of the
Society for Biomaterials, San Diego, CA.
5. MP Olivieri, RM Wollman, EA Mysliwiec-Levendusky and JL Alderfer (1997),
"Conformational Analysis of a Fourteen Amino Acid Mussel Adhesive Protein
Peptide," Abstracts from the Tenth Conversation Issue of the Journal
of Biomolecular Structure and Dynamics, 14, No. 6 Abstract 219.
6. MP Olivieri, RM Wollman and SC Langer (1999) "Biophysical Methods
to Develop and Characterized Potential Bioadhesives" The 25th Annual
Meeting of the Society for Biomaterials, San Diego, CA. Providence,
RI April 27-May 2, 1999.
J.Y. Ostashevsky*
SUNY - Downstate Medical Center,
Box 1212, Brooklyn, NY 11203
*For author correspondence. Phone: (718) 245-2841; Fax: (718) 245-2840 ;
E-mail: ostasj23@hscbklyn.edu
A quantitative model of interphase chromosome higher-order structure is presented, based on the isochore model of the genome and results obtained in the field of copolymer research. G1 chromosomes are approximated in the model as multiblock copolymers of the 30-nm chromatin fiber, which alternately contain two types of 0.5-1 Mbp blocks (R and G minibands) differing in GC content and DNA bound proteins. A G1 chromosome forms a single-chain string of loop clusters (micelles), with each loop ~ 1-2 Mbp in size. The number of ~ 10-20 loops per micelle was estimated from data of Yokota et al. (J. Cell Biol. 130, 1239, 1995) for the dependence of geometrical vs. genomic distances between two points on human chromosome # 4, and from our data (see following abstract) for viscoelastometry of V79 cell lysates. The greater degree of chromatin extension in R vs. G minibands, a difference in the replication time for these minibands (early S phase for R vs. late S phase for G), and the greater amount of inter- and intra-chromosome exchanges in R vs. G minibands are explained in the model as a result of the location of R minibands at micelle cores and G minibands at loop apices. The estimated number of micelles per nucleus is close to the observed number of replication clusters at the onset of S phase. Relationships between chromosomal and nuclear sizes, and between various types of chromosome aberrations are well described in the framework of the model.
Matthew A. Ismail and Alison Rodger
Department of Chemistry,
University of Warwick,
Coventry, CV4 7AL, UK
Self assembly in biological systems is increasingly being recognised as an important phenomenon. We have examined a model system: the cationic meso-substituted free base porphyrin derivative (Figure 1) shown below. It self assembles in solution at the appropriate pH and also self-assembles on DNA. The assembly on DNA is a function of sequence as well as, concentration and mixing ratio, ionic strength, pH and temperature. Resonance light scattering, linear dichroism, circular dichroism, normal absorption and fluorescence spectroscopies have been used to probe DNA/porphyrin systems of different sequence DNAs. In addition, the porphyrin transition moments were analysed with stretched polymer film linear dichroism (LD).
Circular dichroism (CD) and LD data showed a DNAporphyrin binding mode dependence on ionic strength, ligand load on DNA and base composition at the binding site. At low ionic strength and low porphyrin/DNA mixing ratios, exciton effects in the ligand induced CD of all three DNAporphyrin systems indicated the presence of electronic coupling between bound porphyrins. The extent of the coupling increased with porphyrin/DNA mixing ratio and ionic strength. Increasing the ionic strength had the greatest effect on the state of porphyrin aggregation as evidenced by strong resonance light scattering (RLS) in the absorption band of the porphyrin, indicating the formation of extended porphyrin aggregates. Porphyrin aggregation occurred most readily on poly[d(G-C)]2 but was markedly disfavoured in the presence of poly[d(A-T)]2.
For the low ionic strength complex of porphyrin with calf thymus DNA, the average Soret transition moment was determined, from flow LD data, to lie at 4244° to the helix axis. This binding geometry is consistent with groove binding. The porphyrin also binds to poly[d(A-T)]2 in a non-intercalated mode at low ionic strength, as evidenced by positive LD in the Soret region of the LD spectrum. The low ionic strength porphyrin complex with poly[d(G-C)]2 was characterised by both positive and negative LD in the Soret region of the spectrum. This observation is consistent with a binding geometry in which the two Soret transition moments lie above and below 55° to the DNA helix axis. Molecular modelling facilitated interpretation of the spectroscopic data.
Polarised absorbance spectra and average transition moment polarisations were calculated, from LD and UV-visible absorbance data, for the porphyrin oriented in a stretched polymer film.
Figure 1: trans-bis-(4-N -methylpyridiniumyl)diphenylporphyrin.
W. Saenger, P. Orth, C. Kisker, W. Hillen and W. Hinrichs
Freie Universität Berlin,
Institut für Kristallographie,
Takustr. 6,
D-14195 Berlin
and Universität Erlangen,
Institut für Mikrobiologie,
Staudtstr. 5,
D-91058 Erlangen
The family of tetracycline antibiotics act by reversible binding to the
small 30S subunit of procaryotic ribosomes and inhibit protein synthesis.
Since tetracyclines have been heavily used, resistance mechanisms of different
types have emerged. In gram-negative bacteria, the most common mechanism
is due to protein TetA which is located in the bacterial membrane and exports
tetracycline molecules as soon as they have invaded the bacterial cell so
that the antibiotic cannot reach the ribosomal subunits. Since uncontrolled
expression of TetA would lead to unspecific flux of ions across the membrane,
this would destroy the membrane potential and be lethal for the cell. For
this reason, the biosynthesis of TetA is very tightly regulated by the homodimeric
tetracycline repressor (TetR)2 which binds with two helix-turn-helix motifs
to operator DNA (tetO; Kass ~ 11M-1) and blocks
expression of the resistance protein TetA. If tetracycline (Tc) invades
the cell, it chelates Mg2+, and the complex [MgTc]+ binds to and induces a conformational change in (TetR)2 so that its affinity to tetO is reduced by ten
orders of magnitude; expression of TetA can take now place, conferring resistance.
Crystallographic studies show that induction of (TetR)2
by [MgTc]+ is triggered by coordination of Mg2+ to His100Ne, followed by conformational changes of (TetR)2 which involve change of an alpha -helical turn into a
beta -turn. This is associated with pendulum-like movements of helices a
4 and a 4 to which the helix-turn-helix motifs are fixed so that they do
no longer bind to adjacent major grooves in the tetO double helix,
thereby releasing it. The conformation of induced (TetR. [MgTc]+)2 is stabilized by a chain of eight cooperatively bound
water molecules. X-ray studies have revealed all details of tetO
to (TetR)2 binding so that gene regulation by TetR
can be discussed in detail.
A.N. Surovaya1, G. Burckhardt2, S. L. Grokhovsky1, V.
F. Pismensky1, Ch. Zimmer2
and G. Gursky1
1Engelhardt Institute of Molecular Biology,
Russian Academy of Sciences,
Moscow 117984, Russia
2Institute of Molecular Biology Fridrich Schiller
University,
Jena, Germany
Interaction of cis-diammineplatium (II)-bridged bis-netropsin (Nt-Pt(NH3)2-Nt) and oligomethylene-bridged bis-netropsin (Nt-(CH2)5-Nt) with synthetic DNA fragments containing different arrangements of AT base pairs is studied by a combination of CD spectroscopy and fluorescence methods. Netropsin-like fragments were linked in a tail-to-tail manner in two synthetic ligands. It was shown earlier that two ligands bind to DNA and 12-mer DNA oligomer in the extended and hairpin -like conformations (1,2). Binding of bis-netropsins to the following set of DNA oligomers has been studied:
5 '-cctatatcc-3 ' (I) 5 '-ccttattcc-3 ' (II)
3 '-ggatatagg-5 ' 3 '-ggaataagg-3 '
5 '-ccttaatcc-3 ' (III) 5 '-cctttttcc-3 ' (IV)
3 '-ggaattagg-3 ' 3 '-ggaaaaagg-5 '
5 '-ccaatttcc-3 ' (V)
3 '-ggttaaagg-5 '
It was shown that the ligands bind to a 9-mer DNA oligomer with the sequence 5 '-cctatatcc-3 ' (I) only in hairpin form with a stoichiometry 1:1. The existence of partially bound bis-netropsin species containing only one of the two netropsin-like fragments in a bound state was observed in complexes with oligomers II-V. The contents of hairpin and monodentate binding modes in complexes between bis-netropsins and DNA oligomers were calculated by deconvolution of the difference CD spectra. We found that the affinity order for binding of Nt-Pt(NH3)2-Nt in the hairpin conformation is as follows: I > II > III > IV > V. (Nt-(CH2)5-Nt) exhibits the same affinity order, although the equilibrium between monodentate and hairpin binding modes is shifted toward the monodentate binding. For partially bonded monodentate binding mode we observed an opposite order of the binding preferences. Probably the capacity of the TA step to bend DNA towards the major groove thus widening the minor groove permits to accommodate two-stranded hairpin motif and is responsible for this order of binding preferences.
We found that a shorter DNA oligomer with the sequence 5 '-cgtatacg-3 ' can also accommodate the two-stranded parallel peptide motif.
Specific binding of bis-netropsins is determined not only by the nucleotide sequence of the DNA but also depends on the local DNA conformation and bendability at each individual binding site.
Acknowledgment
This work was supported by Deutche Volkswagen Stiftung (grant AZI/70409), Russian Foundation for Basic Research, grant N 96-04-49047 and in part by the State program for Support of the Russian Scientific Schools, N 98093.
References and Footnotes
1. A.N. Surovaya, G. Burckhardt, S.L. Grokhovsky, E.Birch-Hirschfeld,
G.Gursky and Ch. Zimmer, J. Biomol. Struct. Dyn. 14, 595-606 1997.
2. S.L. Grokhovsky, A.N. Surovaya, G. Burckhardt, V.F. Pismensky, B.K. Chernov,
Ch. Zimmer, G.V. Gursky, FEBS Letters 439, 346-350, 1998.
Dmitry Bondarev1 and Carol A. Venanzi2
1Chemistry Department,
Rutgers University,
Newark, NJ 07102
2Department of Chemical Engineering, Chemistry,
and Environmental Science,
New Jersey Institute of Technology,
Newark, NJ 07102
Helical parameters of dinucleotide steps in hexameric and octameric B-DNAs generated by molecular dynamics simulations with the AMBER Cornell, et al. molecular mechanics forcefield are compared to those of the crystal structures of A-type and B-type DNA found in the Rutgers Nucleic Acid Database. The computer-generated structures have sugar pucker distribution similar to crystal B-DNA and different from that of crystal A-DNA. All dinucleotide and base pair steps in the computer-generated fragments have negative sliding and small helical twist values, which makes them more similar to A-type DNA. Base pair steps in the computer-generated fragments are stiffer than those in natural DNAs.
Acknowledgement
This work was supported in part by grants to C.A.V. from the Petroleum Research Fund of the Americal Chemical Society and a generous grant of computing time from the National Center for Supercomputing Applications.
Rolf Misselwitz1,2, Karin Welfle1, Christoph Krafft1, Enrico
Caserta3, Letizia Brandi3,
Cynthia L. Pon3, Claudio O. Gualerzi3 and Heinz Welfle1*
1Max-Delbrück-Centre for Molecular Medicine,
Berlin,
2Institute of Biochemistry,
Medical Faculty (Charité),
Humboldt-University,
Berlin, Germany;
3Laboratory of Genetics,
Department of Biology,
University of Camerino,
Italy
*Author to whom correspondence should be addressed Fax: +49 30 9406 2840.
E-mail: welfle@mdc-berlin.de
CD ellipticity changes at 220 nm show that the fMet-tRNA binding domain (IF2C) of translation initiation factor IF2 unfolds in two steps in the presence of guanidine hydrochloride and indicate that this domain consists of two independent sub-domains having different stability (1). To identify the nature of these two sub-domains, the four Phe residues of IF2C, which are fairly regularly spaced within this domain (i.e. positions 531, 599, 657 and 721), were individually mutagenized to Trp, and the two Cys residues at positions 668 and 714 were exchanged against Val. The resulting variant proteins were overexpressed, purified and characterized.
The fluorescence spectra of the Trp mutant proteins indicate that Trp531 and Trp657 (lambda emiss. max. = 348 nm) are localized on the surface whereas Trp599 and Trp721 (lambda emiss. max. = 332 nm) are buried within the molecule. Spectroscopic studies (CD and Raman) and measurements of the affinity for fMet-tRNA demonstrated that the structural and functional properties of the mutants are almost identical (F531W, F657W and F721W) or very similar (F599W) to those of wt IF2C. When unfolding of the IF2C mutants was followed monitoring Trp fluorescence changes as a function of increasing Gdn/HCl concentrations, the emission maxima were found to change giving rise to one-step unfolding curves with half transitions at 2.6 m Gdn/HCl for IF2C-F721W and at 3.4 m Gdn/HCl for IF2C-F599W. Comparison of these results with those obtained by CD demonstrates that the fluorescence of Trp721 and Trp599 monitors the unfolding of a less stable C-terminal (IF2C-2) and of a more stable N-terminal (IF2C-1) sub-domain of IF2C, respectively.
The Cys>Val mutants have the same CD spectra and undergo the same two-step unfolding in the presence of guanidine hydrochloride as the wild type protein. However, IF2C-Cys668Val was fully active in fMet-tRNAfMet binding while IF2C-Cys714Val was severely impaired in this function. Raman spectroscopy and accessibility studies to thiol reagents indicate that both Cys residues are buried within a hydrophobic core. Therefore, direct participation of Cys714 in the interaction with fMet-tRNA seems unlikely; instead, our data suggest that Cys714 is located in the proximity of the binding site and that the inactivation following its substitution is due to a small conformational change in the neighborhood of the binding site which interferes with the activity of IF2C.
References and Footnotes
1. Misselwitz, R., Welfle, K., Krafft, C., Gualerzi, C.O., & Welfle, H., Biochemistry 36, 3170-3178, 1997.
Erik Winfree*
Department of Molecular Biology,
Princeton University,
Princeton, NJ 08544
and
Computation and Neural Science,
California Institute of Technology,
Pasadena CA 91125
*For author correspondence. Phone: 609-258-4561; Fax: 609-258-2759; E-mail:
winfree@hope.caltech.edu.
Biology makes things far smaller and more complex than anything produced by human engineering. The biotechnology revolution has for the first time given us the tools necessary to consider engineering on the molecular level in DNA computation, launched by Len Adleman, has opened the doorfor experimental study of programmable biochemical reactions. This talk will focus on a single biochemical mechanism, the self-assembly of DNA structures, that is theoretically sufficient for Turing-universal computation. The theory combines Hao Wang's purely mathematical Tiling Problem with the branched DNA constructions of Ned Seeman. In the context of mathematical logic, Wang showed how jigsaw-shaped tiles can be designed to simulate the operation of any Turing Machine. For a biochemical implementation, we will need molecular Wang tiles. DNA molecular structures and intermolecular interactions are particularly amenable to design and are sufficient for the creation of complex molecular objects. The structure of individual molecules can be designed by maximizing desired and minimizing undesired Watson-Crick complementarity. Intermolecular interactions are programmed by the design of sticky ends that determine which molecules associate, and how. This theory has been demonstrated experimentally using a system of synthetic DNA double-crossover molecules that self-assemble into two-dimensional crystals and that have been visualized by atomic force microscopy. This system provides an excellent platform for exploring the relationship between computation and molecular self-assembly, and thus represents a first step toward the ability to program molecular reactions and molecular structures.
Figure 1: A DNA Wang tile with four sticky-end binding domains.
Figure 2: A set of 7 Wang tiles that count in binary.
References and Footnotes
1. Leonard M. Adleman. "Molecular computation of solutions
to combinatorial problems." Science 266, 1021-1024, 1994.
2. Erik Winfree, Furong Liu, Lisa Wenzler, Nadrian C. Seeman. "Design
and self-assembly of two-dimensional DNA crystals." Nature 394,
539-544, 1998.
Igor G. Panyutin1, Valeri N. Karamychev1,2, Ronald D. Neumann1,
Susan Garges3 and Victor B. Zhurkin3
1Warren G. Magnuson Clinical Center,
National Institutes of Health,
10 Center Dr.,
Bldg. 10, Rm. 1C401,
Bethesda, MD 20892-1180
2Novosibirsk Institute of Organic Chemistry,
Lavrenteva 8,
Novosibirsk 630090, Russia
3National Cancer Institute,
National Institutes of Health,
Bldg 12B, Rm. B116,
Bethesda, MD 20892
*Author to whom correspondence should be addressed. Phone: (301) 496-8308;
Fax: (301) 480-9712; E-mail igorp@helix.nih.gov
Frequencies of DNA strand breaks produced by the decay of an Auger electron-emitting
radioisotope such as 125I depend on distances from
the radioisotope to the DNA nucleotides and, therefore, their analysis can
be used to study DNA conformation. To test such radioprobing on a DNA-protein
complex with known crystal structure, we have studied the DNA breaks distribution
in the DNA-CRP complex induced by decay of 125I that
was incorporated in the C5 position of a cytosine in one of the DNA strands.
We found a clear difference in the frequencies of breaks in the DNA-CRP
complex when compared to the naked DNA duplex. This difference correlates
with the increased distances between the deoxyriboses and the radioiodine
atom, caused by the CRP-induced kink in the DNA. Thus, we demonstrated that
radioprobing can detect few angstrom conformational changes of DNA in DNA-protein
complexes in solution.
We also applied radioprobing to study folding of the DNA and RNA strands in the arrested T7 RNA polymerase transcription complex. T7 RNA polymerase transcripts contained 125I-labeled cytosines in different positions relative to the site of the arrest in the series of synthetic DNA templates. We found that the overall yield of the DNA strand breaks is higher in the transcribed than non-transcribed strand. This is consistent with local opening of the DNA duplex and formation of an 8-10 bp hybrid between RNA and the transcribed DNA strand. In addition, we found that the yield of strand breaks in the transcribed strand varies depending upon the position of 125I-C relative to the site of the arrest that suggests a non-uniform structure of the DNA/RNA hybrid. Furthermore, the 125I-induced DNA breaks were detected in both strands "upstream" from the transcription "bubble" which indicates that the DNA and RNA strands remain associated "outside" the RNA polymerase. Thus, our radioprobing data provide a unique insight in the DNA/RNA folding inside transcription elongation complex and demonstrate that this folding is more complicated than had been anticipated in the existing models.
H. Gobind Khorana
Department of Biology,
Massachusetts Institute of Technology,
Cambridge, MA
Rhodopsion, the vertebrate photoreceptor, is a prototypic
molecule in the largest family of G-protein coupled receptors (GPCR). Like
all receptors of this family, it contains three distinct domains: the cytoplasmic
(intracellular) domain that is involved in all the protein-protein interactions;
the transmembrane (TM) domain where the signal transduction begins, by light-catalysed
isomerization of 11-cis -retinal to all trans -retinal; and
the intradiscal domain which appears to be involved in a specific tertiary
structure. The main focus of this talk is to describe efforts to understand
structures and specific functions of the three domains. The main findings
to be presented are as follows: 1. Intradiscal domain contains a globular
tertiary structure. A central feature is a disulfide bond (Cys110-Cys187)
which is conserved in most of the known GPCR. 2. The correct folding in
vivo requires the formation of the above disulfide bond. Misfolding
resulting in non-retinal binding is frequently caused by Retinitis Pigmentosa
(RP) point mutations in the intradiscal and the TM domanin. 3. In vivo
folding studies, using RP mutations in every one of the seven helices, have
been shown that the packing of the helices in the TM diomain and folding
to form the intradiscal tertiary strucure are coupled. 4. Cysteine mutagenesis
has been used systematically to study the tertiary structure and light-dependent
changes throughout the cytoplasmic face by combination of biochemical and
biophysical studies. In particular, EPR spectroscopy following spin labeling
of selected double cysteine mutants has shown movements in helices, including
tilting, following retinal isomerization. 5. Large scale expression of mutants
has allowed application of both 19F-NMR (solution)
and MAS solid state NMR (in collaboration with Dr. Steve Smith's group,
SUNY, Stony Brook). Results of current work are promising for detailed study
of the conformational change. Finally, a unifying hypothesis, which is termed
the central dogma in the GPCR field, will be proposed. This states
that despite the enormous variation in "accessory" structural
details, the principle mechanism of signal transduction starting with pertubation
in the seven helical bundle is fundamentally the same in all GPCRs. Experiments
to test helix movements, the first step in signal transduction following
ligand binding in two adrenergic receptors are now feasible. The patterns
of helix movements in them will be compared with the pattern demonstrated
for rhodopsin and its mutants.
Manju Bansal and Anirban Ghosh
Molecular Biophysics Unit,
Indian Institute of Science,
Bangalore 560012, India
Analysis of available B-DNA type oligomeric crystal structures as well as protein bound DNA fragments indicates that AA.TT and GA.TC dinucleotide steps, show remarkable resistance to change, even when occurring in protein bound structures. In addition the electropositive C2-H2 group of adenine is in very close proximity of the electronegative carbonyl oxygens of pyrimidine bases in the antiparallel strand of the duplex structures, suggesting the possibility of both intra-basepair as well as cross-strand C-H..O hydrogen bonds in the minor groove. The donor group is the C2H2 of the 3'-adenine in both sequences, while the O2 acceptor is from the 3'-thymine in case of AA.TT and 3'-cytosine in the case of GA.TC steps. The C2-H2..O2 hydrogen bonds in the A.T base pairs could be a natural consequence of Watson-Crick pairing. However, the cross-strand interactions obviously arise due to local sequence dependent geometry of the AA.TT and GA.TC steps, since a majority of the H2..O2 and C2..O2 distances are considerably shorter than their values in a uniform fibre model, and also closer than the sum of their van der Waals' radii, in both data sets. Since the GA.TC step does not generally have large propeller twist, nor does it have exocyclic groups capable of favourable cross-strand interactions in the major groove (in fact the interaction between the O6..O6 groups will be strongly repulsive) it appears that, the C-H..O cross-strand interactions in the minor groove play an important role in determining the structural rigidity of individual AA.TT, GA.TC steps, and also An.Tn and GAn.TnC tracts in DNA olgomer, as well as protein bound complexes. In addition, since similar interactions are seen in structures with specific hydration patterns in the minor groove, as well as those without such solvent interactions, it seems reasonable to assume that the cross-strand C-H..O hydrogen bonds are an intrinsic feature of AA.TT and GA.TC steps, which lead to dinucleotide doublet geometries that give rise to a narrow minor groove when such sequences occur in consecutive steps. This then provides an ideal scaffold for the formation of a spine of solvent in the minor groove, as seen in many crystal structures or binding of drug molecules. It is also interesting to note that the same features which lead to a narrow minor groove also lead to the major groove becoming deeper so as to accommodate a protein alpha helix.
Helen M. Berman, John Westbrook, Kyle Burkhardt, Zukang Feng, Shri
Jain, Rachel Kramer, Bohdan Schneider and Christine Zardecki
Rutgers University,
Piscataway, NJ
Peter Arzberger, Phil Bourne, John Badger and Helge Weissig
San Diego Supercomputer Center,
University of California, San Diego,
La Jolla, CA
Gary Gilliland, Phoebe Fagan, Hillary Gilson, Diane Hancock, Narmada
Thanki and Greg Vasquez
National Institute of Standards and Technology,
Gaithersburg, MD
On October 1, 1998, the Research Collaboratory for Structural Bioinformatics (RCSB) became responsible for the management of the PDB. The RCSB members are Rutgers, the State University of New Jersey, the San Diego Supercomputer Center of the University of California, San Diego, and the National Institute of Standards and Technology.
The vision of the RCSB (http://www.rcsb.org/) is to enable new science by providing accurate, consistent, and well-annotated structure data via the application and development of modern information technology.
Data is deposited and processed by the RCSB using an integrated data processing system called ADIT (the AutoDep Input Tool). ADIT provides rapid and reliable data processing, and is also being used to revisit all existing structures in the PDB to create a more uniform archive.
The RCSB has also developed a query and reporting interface to search across the PDB archive. Searches and reports can be generated for single or multiple structures. As the quality of the data improves, the reliability of the query results will improve.
These systems and plans for extending the capabilities of the new PDB will be described.
This project is funded by the National Science Foundation, the Department of Energy, and two units of the National Institutes Of Health: the National Institute Of General Medical Sciences and the National Library Of Medicine.
Karl D. Bishop1,2, Lee Bickerstaff1, Elizabeth Lozada1 and
Kim Dunbar
1Department of Chemistry,
Michigan State University,
East Lansing, MI 48824
2Department of Chemistry,
Bucknell University,
Lewisburg, PA 17837
Since the discovery that cisplatin and other platinum [Pt(II) and Pt(IV)] complexes inhibit DNA replication, much effort has been expended in designing and structurally characterizing model compounds for eventual use as chemotherapeutic agents. The search for non-platinum based complexes
has intensified in recent years due to the high toxicity of the Pt compounds to the patient and the ineffectiveness of the compounds on certain types of cancers (head, neck, ovarian and testicular). We are therefore investigating the sequence specificity and structural characteristics of other metal compounds and their role as DNA binding ligands. Among the compounds which exhibit carcinostatic activity are dinuclear complexes of rhenium, rubidium and rhodium [Re2(III,III), Ru2(II,III) and Rh2(II,II)]. Recent studies have found that these dinuclear compounds react with guanine to produce a bridging interaction that has not been observed before with DNA and mononuclear metal complexes (Day et al., JACS, 116 , 2201 (1994)). The dimetal/DNA structure is bent by ~90 at the ligand binding site.This modeling study, which demonstrates severe structural distortions, is corroborated by NMR results. Mononuclear metal binding to DNA also results in structural distortion, but not to the extreme degree exhibited here. This suggests a structural basis for the different biological activitiesof the metal compounds.
NMR data acquisition and analysis on the ligand free duplex and the complex is progressing very well. We have assigned the majority of the exchangeable and nonexchangeable proton resonances for the DNA duplex. NMR based restrained molecular dynamics of the ligand-free duplex is in progress. NMR analysis of the ligand-DNA complex has begun and we are in the process of determining the optimum experimental conditions. Our results show that the duplex is destabilized by the addition of dirhodium biscarboxylate, but still forms hydrogen bonded base pairs, allowing us to accomplish the NOESY walk along one strand.
We have demonstrated the ability of rhodium bisacetate to bind to double stranded plasmid DNA. Rhodium bisacetate of varying concentrations (4.5mM-23nM) were incubated overnight with 1ug double stranded plasmid pBR322. Samples, along with a no-metal control, were separated by electrophoresis on 1% agarose, stained with ethidium bromide and photographed. The profound mobility shift changes of the supercoiled plasmid at high dimetal concentration are indicative of unwinding of the DNA sequence, causing significant disruption and destabilization of the plasmid structure. Similar disruption is evident in both the closed circular and nicked forms of the plasmid. A second, similar study has demonstrated the ability of rhodium bisacetate to interrupt polymerase activity. pBR322 was incubated with varying concentrations of rhodium bisacetate. With the addition of radiolabelled primer, Taq polymerase and the appropriate buffer components, the samples underwent PCR. Inhibition of PCR in certain samples indicates inhibation of polymerase activity by the dimetal. Both studies represent the first conclusive evidence that rhodium bisacetate does bind to double stranded DNA.
We have recently begun investigations to determine the binding kinetics
of rhodium bisacetate to DNA. Properties under investigation include preferential
binding site(s), reaction rate, temperature, and complex stability. Mass
spectroscopy, HPLC and NMR, along with classical biochemical techniques,
are being employed in these studies.
Charles R. Cantor
Sequenom Inc,
San Diego CA and Sequenom GmbH,
Hamburg, DE
Sequenom has developed the technology to scan an array of samples in a chip format by Matrix Assisted Laser Desorption Ionization (MALDI) time of flight (TOF) Mass Spectrometry (MS). Current instrumentation uses samples that are about 200 microns square. For DNA samples about 6 fmol of material is required; proteins and other analytes would require far less material. Mass spectra can be acquired automatically at a rate of about 3.5 seconds per sample. Apparatus now available commercially can process ten 384-sample chips simultaneously. Chips pre-filled with matrix will also be available soon. Initially, matrices for DNA samples will be offered, while matrices suitable for other applications will be developed later.
DNA analysis by Sequenom's MassArray technology covers a gamut of applications from sequencing and allele detection to mutation finding and gene expression analysis. Allele determination is the most well developed application and the one likely to see large-scale adoption in the near term. Current MS technology limits the size of DNA samples examined to around 60 to 80 bases. Thus PCR must be used to subdivide a larger target into fragments in this size range. Advances in IR MALDI are ongoing and these should eventually allow the size range of high resolution DNA MALDI MS to be extended by two to four fold. MS analysis of DNA offers several advantages over more conventional methods. Because of the much higher size resolution of MS compared with electrophoresis, many targets can be processed in a single spectrum. Such a procedure is called multiplexing. In MS DNA sequencing, the high resolution allows unequivocal detection of heterozygotes and in some cases even allows the phase of compound heterozygotes to be called. This last fact is very important in clinical diagnostics. All other methods in current use require prior sub-cloning for phase determination. This is feasible only in a research setting.
Jannette Carey
Chemistry Department,
Princeton University,
Princeton, NJ 08544-1003
In order to carry out their biological functions, many DNA-binding proteins must be able to recognize a range of similar but non-identical DNA sequences while maintaining the ability to reject slightly more distantly related sequences. Thus, these proteins must execute a delicate balance between affinity and specificity. The molecular and biological mechanisms used to achieve this balance will be explored using the examples of the tryptophan and arginine repressor proteins.
P. Acharaya, A. Trifonova, C. Thibaudeau and J. Chattopadhyaya*
Department of Bioorganic Chemistry,
Box 581, Biomedical Centre,
University of Uppsala,
S751 23 Uppsala, Sweden
The intrinsic dynamics and architectural flexibility of nucleic acid resulting in to specific function are the result of cooperative interplay of pentofuranose, nucleobase and phosphodiester moieties. We have earlier shown [see http://bioorgchem.boc.uu.se/] that the interplay of various stereoelectronic gauche and anomeric effects and associated steric effects energetically drives the sugar conformation, which in turn is dictated by the electronic nature of the aglycone and other substituents on the sugar ring.
In this work, we have employed guanosine 3',5'-bis-ethylphosphate as a model mimicking the central nucleotide moiety in a trinucleoside diphosphate in order to shed light on how the strength of intramolecular stereoelectronic effects is modulated by the change of protonation-deprotonation equilibrium in complete absence of any intramolecular base-base stacking. The work uniquely shows a complete interdependency of conformational preference of sugar and phosphate backbone in ribonucleoside 3'-ethylphophate as the of protonation-deprotonation equilibrium of the aglycone changes as a function of pH.
C.R. Grace, Andrew Lynn and Sudha M. Cowsik
School of Life Sciences,
Jawaharlal Nehru University,
New Delhi 110 067, India
Phone 91 11 617735; Fax: 91 11 6187338; E.mail scowsik @ hotmail.com
Sophisticated Instruments Facility,
Indian Institute of Science,
Bangalore 560012, India
Phone 91 80 3092343; E.mail grace @ sif.iisc.ernet.in
The tachykinin family neuropeptides is characterised by the C-terminal pentapeptide sequence Phe-X-Gly-Leu-Met-NH2 (X=an aromatic or aliphatic residue). These peptides show a wide spectrum of biological actions in the central and peripheral nervous system such as these excite neurons, evoke behavioral responses, are potent vasodilators and contract many smooth muscles. The possibility that tachykinins may act as growth factors or as messengers between the nervous and immune system has generated much interest and excitement.
To better understand the structural basis of the biological activity of tachykinin, kassinin, a 12 residue peptide of amphibian origin, the two dimensional NMR spectroscopy experiments and stimulated annealing calculations have been used to investigate the conformation adopted in the presence of membrane model system. Circular dichroism spectropolarimetry has been used to evaluate the effect of solvent on peptide folding. These data show that in solution, kassinin exists in a mixture of conformational states rather than a single three dimensional structure. In water, Kassinin, prefers to be in an extended chain structure. In the presence of membrane mimetic solvents, a helical structure is induced in the C-terminus segment. This is expected to provide a possible structure of neurokinin-2 receptor selective ligands.
C.W. Hilbers, M.H. Kolk, P.J.A. Michiels, S.S. Wijmenga*, C.W.A. Pleij
and H.A. Heus
NSR Centre for Molecular Structure, Design and Synthesis,
Laboratory of Biophysical Chemistry,
University of Nijmegen,
The Netherlands
Leiden Institute of Chemistry,
University of Leiden,
Leiden, The Netherlands
*Present address:
University of Umeå,
Sweden
New structures of RNA moleculaes appear in a rapid pace revealing many unexpected new folding motifs that underscore the structural versatility of RNA. In my presentation some of these developments will be discussed on the basis of the structure of the pseudoknot occurring at the 3'-end of the Turnip Yellow Mosaic Virus (TYMV) RNA as well as of other and related hairpins, e.g. the ribozyme substrate hairpin of Neurospora VS RNA.
The TYMV molecule is rodshaped and consists of three stem regions which are on average co-axially stacked. The loops which span the major and minor groove of the core helices of the pseudoknot exhibit unexpected structures. Furthermore, it turned out that the conformation of an isolated part of the molecule is remarkebly similar to the corresponding region in the pseudoknot. This implies that it is preformed for folding into a pseudoknotted structure. The NMR data show that the molecule does not behave as a solid object in solution, but that there is significant flexibility between the helical subdomains. The corresponding motions occur in the millisecond domain. Conformational exchange on a millisecond time scale was also found in other examples a wide spread phenomenon in RNA.
An-Suei Yang and Barry Honig
Department of Biochemistry and Molecular Biophysics,
Columbia University,
630 W. 168 St., New York, NY 10032
In the next few years the increasing availability of three-dimensionalstructural information will have major impact on the ability to exploit genomic information. PrISM, an integrated sequence/structural-analysis/homology model building program will be introduced. The program consists of a variety of linked modules which include the facility to carry out sequence analysis, structure-based sequence alignment, fast structure-structure superposition using a unique structural domain database, multiple structure alignment, threading and homology model building. Some of the features and applications of the program will be described. These include unique structure-based sequence profiles for protein families, new relationships between measures of protein and sequence similarity, and results from automated homology model-building. PrISM was used, with a considerable level of success, to make predictions for all 43 targets at the recent CASP3 experiment. The current state of protein structure prediction and likely future developments will be discussed in light of current structural genomics initiatives.
Laura Landweber*
Department of Ecology & Evolutionary Biology.
Princeton University,
323/328 Guyot Hall
Princeton, NJ 08544-1003
http://www.princeton.edu/~lfl
*For author correspondence. Phone: 609-258-1947 ; Fax: 609-258-1682; E-mail:
lfl@princeton.edu
We have expanded the field of "DNA Computers" to RNA to solve a class of mathematical problems in chess. The versatility of RNA makes it even more suitable than DNA for molecular computation. For example, 'bit'-operations can be performed on RNA using specific RNase, ribozyme, or deoxyribozyme digestion of target RNA molecules, leading to the construction of a purely nucleic acid "computer".
I will demonstrate a solution to the so-called "Knight problem" which actually has many solutions, like problems in biology. Simply put, this problem asks how many knights (or kings or queens etc.) and in what configuration can one stably place them on an N x N chess board such that no knight is attacking any other knight on the board. This problem can be solved using only biological molecules and enzymes as tools. For a simple case like a 3 x 3 chess board, the 9 spaces on the chess board correspond to 9 'bits' or place-holders in an RNA sequence. A bit flipped to 'on' or 1 represents a knight at that position, and 'off' or 0 means that position is empty. Each round of selection, performing single bit-operations, destroys only those RNA molecules that do not satisfy a single criterion for this problem. The set of solutions can be read by either RT-PCR screening, RNase H digestion, or direct sequencing of the 'winning' molecules.
Ingrid Lafontaine and Richard Lavery
Laboratoire de Biochimie Théorique, CNRS UPR 9080
Institut de Biologie Phjysico-Chimique
13 rue Pierre et Marie Curie, Paris 75005, France
Theoretical studies of the effects of base sequence on either nucleic acid conformation or interactions all run into one fundamental problem. Although there are only four standard bases in DNA, the number of combinations which can be made from these bases increases exponentially with the length of the sequence. For a segment of 10 base pairs there are already more than a million possibilities and for 15 base pairs the number rises to 109. This implies that if we want to find the best sequence for binding a given protein, we would have to try out millions of possibilities and the same is true if we ask a biotechnologically more important question - what effect will a protein mutation have on its optimal target sequence?
Experimentally, such questions have been addressed using the SELEX approach (1) via combinatorial libraries containing millions of oligonucleotide sequences. The same technique has also led to the creation of DNA and RNA aptamers which are able to selectively bind chosen target molecules. We propose to adapt this approach to the computer by a novel use of mean field techniques. Our methodology, based initially on the JUMNA (2) internal coordinate molecular modeling approach, uses nucleotides containing all four standard bases. The contribution of each of these bases to the energy is controlled by coefficients which can vary during minimization. In this way, we are able to optimize nucleic acid fragments both with respect to their conformation and with respect to their sequence. This allows us to use the computer to solve problems of the type studied with SELEX, but also allows us to go beyond the limits of the experimental approach and design sequences with, for example, given mechanical properties.
References and Footnotes
1. Tuerk, C. & Gold, L., Science 249, 505-510,
1990.
2. Lavery, R., Zakrzewska, K. & Sklenar, H., Comp. Phys. Commun.
91, 135-158, 1995.
Jean-Marc Malinge, Franck Coste, Charles Zelwer and Marc Leng
Centre de Biophysique Moléculaire, CNRS
Rue Charles Sadron,
45071 Orléans cedex2, France
The distortions induced in DNA by a cisplatin (cis-diamminedichloroplatinum(II)) interstrand cross-link have been previously characterized in solution by means of several techniques: Gel electrophoresis, chemical probes and 2D 1H nuclear magnetic resonance. We now report the crystal structure of a 10 base pairs double-stranded oligodeoxyribonucleotide containing a single site specific interstrand cross-link resulting from the chelation of the N7 position of two guanine residues on the opposite strands at the d(GpC).d(GpC) site by the cis-diammineplatinum(II) residue. The crystals allow diffraction beyond 1.6Å resolution. The crystal structure was solved at 100K using the anomalous scattering (MAD) of platinum as an unique source of phase information. Several features are in good agreement with the results obtained in solution such as the extrusion of the cytosine residues, the position of the platinum residue in the minor groove, the direction of the bending towards the minor groove and the large unwinding of the double helix. Hydration of the double helix at the platinum site is now determined. Two water molecules are located on either side of the square plane of the platinum residue at about 3.6 è from the platinum along its quaternary axis.These two water molecules, seven other water molecules, the two NH3 ligands of platinum and the two O6 of the cross-linked guanines form a well-defined cage embeding the platinum residue. The cage is linked by other water molecules to the phosphate groups of the cross-linked guanine residues and participate to the widening of the minor groove. On the other hand, the water molecules in apical position with respect to the square plane of the platinum residue can participate to the chemical instability of the cisplatin interstrand cross-links. The bonds between platinum and the N7 of guanine residues are spontaneously cleaved with essentially one cleavage per cross-linked duplex in either of both strands.
Robert T. Sauer, Matthew H.J. Cordes, Nathan P. Walsh and C. James
McKnight
Department of Biology,
MIT,
Cambridge, MA 02139
and Department of Biophysics,
Boston University Medical School,
Boston, MA 02118
A mutant of the P22 Arc repressor homodimer was constructed by switching the sequence positions of a residues in the hydrophobic core and an adjacent residue on the protein surface. The mutant adopts a novel fold in which the beta-sheet present in the wild-type protein is replaced with a right-handed helix. Dramatic repacking of the hydrophobic core also occurs in the mutant. The induced structural changes allow the mutant protein to maintain optimal burial of hydrophobic surface. A mutant with just one of the two "switch" mutations can adopt both the wild-type and the mutant structures and shift between them on the millisecond time scale.
Dino Moras and Jean-Marie Wurtz
Laboratoire de Biologie Structurale, IGBMC
1 rue L. Fries BP163
67404 Illkirch Cedex - France
Nuclear hormone receptors constitute a superfamily of ligand-dependent transcription factors. These molecules display a modular structure with two conserved regions: the DNA binding domain (DBD) which presents the highest degree of sequence conservation and the ligand binding domain (LBD) which contains a ligand dependent transactivation function (AF-2). LBD's level of sequence conservation is significantly lower and in vivo they act as homo or heterodimers.
The crystal structures of the LBDs of RXRa and RARg with and without bound ligands provided a key for our understanding of various structural and functional aspects. Topics such as the origin of the specificity towards ligands and the mechanism of ligand induced transactivation could then be addressed at the atomic level. The position of helix 12 which contains the AF-2 domain is correlated to the transactivation hability of the nuclear receptors. The molecular mechanisms involved in this ligand dependent function are now understood. The presentation will focuse on the following problems: ligand-protein interactions, specificity, molecular mechanisms of agonism and antagonism.
E. N. Moudrianakis
Biology Department,
Johns Hopkins University,
Baltimore, MD, 21218
We have been investigating the types of physicochemical rules that govern the formation of the architectural states of chromosomal nucleoproteins. First, we analyzed the potential of the DNA double helix, in the absence of any other macromolecules, to assume biologically-meaningful compact states. We found that modulation of the water activity in the microenvironment of the helix facilitated the formation of chromatin-like, left-handed supercoils (1). Core histones, in vitro, organize themselves into oligomeric states which upon binding with the double helix drive the resulting nucleoprotein complex (chromatin) into similar left-handed supercoils with a beaded and discontinuous morphology (2,3). We concluded that the histone octamer drives the wrapping of the double helix around the outer circumference of the protein core by striping away water from (and/or, fixing it towards) the apposing DNA surface and thus the helix collapses directionally for steric and electrostatic reasons. We have solved the crystal structure of the core histone octamer (4) and have modeled the DNA around it (5) with atomic detail. The stereochemistry of the octamer surface and that of DNA proved sufficient to yield a unique solution to this protein-DNA docking problem which was later found in agreement with the more recent results of Luger et al (6). A central protein scaffold, the histone fold (4,7) has been found to provide the fundamental framework for the organization of the protein spool around which the DNA wraps. The four self-assembling protein units are heterodimers formed by the antiparallel association of two histone folds via the handshake motif (4,7) of assembly. The nucleosomal-surface sites from which the histone amino termini emanate have been mapped (5). They are spaced at regular intervals around the DNA trajectory within the nucleosome and periodically alternate their emergence on the sides of the DNA gyres. Their occurrence provides architectural clues for the next level of chromatin organization.
More recently, and in collaboration with Drs. C. S. Tung and A. E. Garcia of Los Alamos, we have completed molecular dynamics simulations of the nucleosomal system in 0.14M NaCl (excess salt) under periodic boundary conditions, at room temperature and for a period of 1.2 nanoseconds. We find that the hydrated nucleosome is an extremely dynamic system where the DNA atom motions dominate those of the protein by a factor of ca. 10. Base pair wedges form and disappear in this time-scale and are dependent on arginine side-chains bridging phosphates across the minor groove of DNA. Further, we find that the nucleosome is surrounded by a cloud of positive ions with density 5-10 X that of the bulk concentration, and that water is also localized at the protein-DNA interface, within the DNA grooves and, especially the apposing DNA gyres. We will attempt to integrate these findings in the context of chromatin physiology.
References and Footnotes
1. Cell, 13, 295, 1978.
2. Biochemistry, 17, 4955, 1978.
3. Cell, 4, 281, 1975.
4. Proc. Natl. Acad.Sci., 88, 10148, 1991.
5. Proc. Natl. Acad. Sci., 90, 10489, 1993.
6. Nature, 389, 215, 1997.
7. Proc. Natl. Acad. Sci., 92, 11170, 1995.
8. Cold Spring Harbor Symposia, LVIII, 275, 1993.
T. Nguyen, I. Rouzina and B. I. Shklovskii
Theoretical Physics Institute,
University of Minnesota,
116 Church Street Southeast,
Minneapolis, Minnesota 55455
Highly charged polymer or a membrane in solution is screened by counterions. Its incomplete screening results in the long range repulsion between distant parts of the macromolecular surface. Therefore, intrinsic rigidity of the membrane, k 0, increases by the salt dependent value k el, k = k 0 + k el. This picture holds for screening by monovalent counterions.
As was shown recently (1), in case of screening with multivalent counterions, the later form on the surface a two-dimensional, (2D), layer. Mutual repulsion between counterions in this layer results in the strong correlation of their positions. If correlation energy per ion epsilon, ref. (2)
is large compared to ion's thermal energy k B T, i.e. traditionally used parameter G = 0.9 epsilon /k B T >>1, the system appears to be quite similar to the 2D Wigner crystal, (WC). Here e is unit electron charge, D = 80 is the dielectric constant of water, a = n -1/2 is average distance between ions along the surface with the uniform charge density s and the ion's 2D number density n = s /Ze.
Under these circumstances, each counterion acquires additional energy of binding ~ epsilon, such that practically all neutralizing ions are bound right at the surface (3). Therefore positive electrostatic contribution to macroion's rigidity vanishes.
In this study we show that formation of ionic WC at the surface leads to the negative electrostatic contribution k el = < 0 to the membrane's rigidity:
where d is the membrane's thickness.
Expression (2) is exact in the limit of large G, i.e. in the case of high surface charge density and high counterion charges. But extensive Monte Carlo simulations (4) show, that even moderate G > 5 values result in the strongly correlated 2D liquid, similar in thermidynamic properties to the perfect 2D WC.
Therefore ion correlations play an important role for the biologically relevant values 5 < G < 15. For example, for the macromolecular surface charge density le per 100Å 2 typical for DNA and many membranes screened with mono-, di-, tri or tetra- valent ions G = 1.41, 4.00, 7.33, 11.28 respectively. Therefore negative contribution to k, has its full strength eq. (2), for Z = 3 and 4, is about twice reduced for Z = 2, and vanishes for Z = 1.
According to expression (2) for 20Å thick membrane charged to le per 100Å 2 and screened with Z = 2,3 and 4 valent counterions k el = -2.1Z 1/2 = -2.97, -3.64 and 4.2 k B T respectively. Such |k el | is significant when compared to the rigidity of the typical lipid membrane with k 0 ~ 10k B T.
Similarly, in case of the charged polymer with the average surface charge density s and diameter d, such that pi d > a, there is analogous negative ion correlation contribution to polymer's persistence, L, similar to eq. (2):
For a polymer like DNA with d = 20Å and uniform surface charge density, L el = 33.2Z 1/2Å, i.e. L el = -47; -57.5 and 66.4Å for Z = 2 3 and 4 respectively. These values should be compared to the chemical persistence length of B-DNA L 0 = 500Å.
Many experiments show significant increase in flexibility of B-DNA in the presence of multivalent cations, such as Mg 2+, Ca 2+, CoHex 3+, polymines, which bind to doublehelix in the nonspecific electrostatic manner. Recently this effect was characterized quantitatively, in DNA stretching experiments with optical tweezers (5), which showed decrease in DNA persistence length to as low as 250-300Å. Present calculations show that strong correlations of multivalent counterions on DNA surface can account for a significant portion of this decrease. The rest of the persistence length reduction results probably from the "permanent bends" in doublehelix, induced by mulitivalent cations captured in DNA grooves (5).
References and Footnotes
1. I. Rouzina, V. A. Bloomfield, J. Phys. Chem. 100,
9977, 1996.
2. L. Bonsall, A. A. Naradudin, Phys. Rev. B15, 1959, 1977.
3. V. I. Perel and B. I. Shklovskii, cond-mat/9902016.
4. H. Totsuji, Phys. Rev. A 17, 399, 1978; F. Lado, Phys. Rev. B17,
2827, 1978.
Sylvie Nonin* and Jean Louis Leroy
*Author to whom correspondence should be addressed. Phone: (33) 1 69 33
44 13; Fax: (33) 1 69 33 30 04; E-mail: sn@pmc.polytechnique.fr
Repetitive DNA sequences may adopt unusal pairing arrangements leading to structures such as G-tetrads or i-motifs for G-rich and C-rich strands respectively. For example, a part of the C-rich CENP-B box of satellite a, one of the seven human centromeric satellites, adopts an i-motif structure (1). The human satellite III, which includes the tandem repeats (CCATT)n.(GGTAA)n (2), could be part of the functional centromer (3) like satellite a. The G-rich strand can form stem-loop structures in vitro (4,5,6). Its C-rich counterpart has been shown to be mostly structureless above pH 7 (7), but as yet no structural studies have been carried out at pH<7.
At acid to neutral pH, many cytosine-rich DNA oligomers form the i-motif structure in which two parallel duplexes with C.C+ pairs are intercalated head-to-tail (8). The i-motif may be formed by multimeric associations or by intra-molecular folding, depending on the number of cytosine tracts, the nucleotide sequence between them, and the experimental conditions such as strand and salt concentrations.
We found that d(CCATT)3CC and several natural derivatives (i.e. oligonucleotides containing natural substitutions such as occur in satellite III) fold into i-motif structures. At the NMR concentration of 1 mM, most of these C-rich oligonucleotides display a multiplicity of structures and stoichiometries, in pH-dependent proportions. This sharply limits any structural study.
On the other hand, two sequences yielded good spectra: d(mCCATTCCATTCCTTTCC) and d(mCCTTTCCATTCCATTCC). The substitution TTT for the ATT spacer occurs naturally in satellite III. Both oligonucleotides and the uridine derivative d(mCCATTCCAUTCCUTTCC) display similar NMR features and responses to experimental conditions. They exhibit i-motif structures with two different intercalation modes depending on pH. At pH 6, the structure is a monomeric i-motif with the following base-pair intercalation: mC1.C11+ / C7.C17+ / C2.C12+ / C6.C16+. The structure is stable up to pH 7 at 0°C. Molecular modelling derived from NMR constraints was carried out for the uridine derivative whose spectra are better resolved. The C.C+ pairs form an i-motif core, while the interspacing segments are looped. The first ATT and the UTT tracts loop over the two narrow grooves of the core. They are connected by a reverse Watson-Crick A3.U13 pair, which extends the i-motif core beyond the C6.C16+ pair. The dissociation constant of this pair, derived from imino proton exchange measurements, is around 5 10-2 at 0°C. The middle ATT segment lies in the major groove, atop the mC1.C11+ base pair. It displays a structure never seen before within monomeric i-motif structures, in which none of the residues stack on the terminal mC1.C11+ pair: A8 is flipped in the major groove coming close to the mC1 and C7 residues, while U9 and T10 loop out with their imino protons pointing outwards. This may explain why the mC1.C11+ pair has a shorter lifetime (2 ms at 0°C) than C7.C17+ (700 ms), C2.C12+ (>1 mn) and C6.C16+ (215 ms), as measured by their imino proton exchange time.
The observation that both the C-rich and the G-rich strands of the human satellite III tandem repeats can fold into compact structures with loops available for ligand recognition may be relevant to the role of the centromer during mitosis and meiosis.
References and Footnotes
1. Gallego et al., J. Mol. Biol., 273, 840-856,
1997.
2. Prosser et al., J. Mol. Biol., 187, 145-155, 1986.
3. Gradi et al., P.N.A.S. USA 89, 1695-1699, 1992.
4. Chou et al., J. Mol. Biol., 259, 445-457, 1996.
5. Jaishree and Wang, FEBS Letters, 347, 99-103, 1994.
6. Castati et al., Biochemistry , 33, 3819-3830, 1994.
7. Gupta et al, Structural Biology: The State of the Art. Proceedings of
the Eight Conversation, State Univ. of New York, Albany, NY 1993, Eds. Sarma
and Sarma, Adenine Press 1994, 137-154.
8. Gehring et al, Nature , 363, 561-565, 1993.
Guillaume Bertucat, Richard Lavery and Chantal Prévost
Laboratoire de Biochimie Théorique, CNRS UPR 9080,
Institut de Biologie Physico-Chimique,
75005 Paris - France
We have recently proposed an atomic scale model for DNA strand exchange
promoted by RecA, based on DNA modeling and available experimental data
(Bertucat, Lavery and Prévost, J. Biomol. Struct. Dynam. 16,
535-546 (1998); Bertucat, Lavery and Prévost, submitted to Biophys.
J.). RecA promoted strand exchange begins with the uptake of a homologous
duplex DNA by the nucleoprotein filament formed by a DNA single strand and
RecA. In this process, the duplex undergoes stretching and unwinding deformations.
We have shown that these deformations allow single strand association via
the minor groove, resulting in the formation of a parallel triple helix.
Our recent calculations strongly suggest that this triplex is the starting
point for strand exchange, spontaneously rearranging via base pair switching
into a more classical triplex, where the single strand occupies the major
groove of the newly formed heteroduplex. This model is supported by various
experimental results and its implications regarding the RecA filament rationalize
various structural and biochemical observations. How homologous recognition
takes place within this mechanism however remains to be determined. We present
here the results of an extensive investigation of the effects of heterology
introduction on both triplex stability and on the strand exchange reaction.
These results support the multi-step recognition process proposed by Bazemore
et al. (Proc. Natl. Acad. Sci. USA 94, 11863-11868) and imply different
levels of discrimination depending on the nature of the mismatch.
Qiang Zhao1, Trixie Wagner1,
Sepideh Khorasanizadeh1, Paul B. Sigler2, Mitchell A. Lazar3 and
Fraydoon Rastinejad1
1Department of Pharmacology,
X-ray Crystallography Laboratory,
University of Virginia,
Box 448,
Charlottesville, VA 22908
2Division of Endocrinology, Diabetes, and Metabolism,
Department of Medicine,
Genetics and Pharmacology,
University of Pennsylvania,
Philadelphia, PA 19104
3Howard Hughes Medical Institute,
Yale University,
Dept. of Molecular Biophysics and Biochemistry,
New Haven, CT 06510
The nuclear receptors form the largest known family of transcription factors, with more than 150 members. Most receptors regulate gene expression through DNA response elements containing the same consensus six base pair half-site. The binding site repertoire is created through various bipartite arrangements of the half-site and receptor homo- and heterodimerization. The retinoid X receptor (RXR) acts as a common dimerization partner for many receptors, enabling their cooperative assembly on specific response elements. To provide a stereochemical understanding for DNA recognition and subunit assembly, we have solved crystal structures of several different receptor-DNA complexes, each involving a homodimer or heterodimer of receptor DNA-binding domains bound to their idealized target elements.
The 2.1 crystal structure of the RXR DNA-binding domain (DBD) on a direct repeat of half-sites spaced by one base-pair (DR1) shows a different dimerization interface than the 1.7 structure of the RXR-RAR heterodimer DNA-binding complex on the same DR1 site. In each case, however, the dimerization interfaces are stabilized by the minor groove contacts with the spacer region. The RXR-RAR complex shows how the dimer interface restricts the subunit polarity, preferentially placing RXR downstream and RAR at the upstream position. The heterodimerization surface simultaneously pulls each receptor into optimal alignment with the half-sites.
We have also solved the 2.3 resolution crystal structure of the DNA binding
region of the orphan receptor RevErb arranged as an asymmetric homodimer
on its target, which is a DR2 response element. This structure, like the
RXR-TR complex (1.9 resolution) reveals the presence of a second major protein-DNA
interface adjacent to the classical one encoded by the C-terminal extension
of the DBD. In each case, the "second" interface allows the necessary
response element selectivity and simultaneously helps establish the dimerization
interface. Collectively, the four structures completed to date show a common
mechanism by which the spacing between the half-sites confers the precise
geometry necessary for subunit dimerization, and aligns the flexible protein
surfaces to make optimal allosteric contacts within the complex.
Thomas Schwartz1, Mark A. Rould2, Ky Lowenhaupt1, Alan Herbert1 and Alexander Rich1*
1Department of Biology,
Massachusetts Institute of Technology,
Cambridge, MA 02139
2Department of Molecular Physiology and Biophysics,
University of Vermont,
Burlington, VT 05405
*Author to whom correspondence should be addressed. Fax: (617)253-8699;
E-mail: cbeckman@mit.edu
Zalpha is a highly specific Z-DNA binding domain, identified from the
N terminus of the editing enzyme double-stranded RNA adenosine deaminase
(ADAR1). We have determined the crystal structure of this protein bound
to a 6 base pair segment of Z-DNA, refined at 2.1 Å resolution. Although
Zalpha is a helix-turn-helix (HTH) protein, it utilizes this common fold
in a way that is strikingly different than that seen with B-DNA substrates.
The specificity of Zalpha for the Z conformation of DNA can be explained
because DNA recognition is mediated primarily via numerous contacts with
the 'zig-zag' sugar-phosphate backbone. The binding surface of Zalpha is
structurally complementary in both shape and electrostatic nature to a stretch
of 5 phosphates. Further, Zalpha contacts a guanine base in the syn
conformation, another hallmark feature of Z-DNA. Because any base in the
syn conformation could make this contact, it is not surprising that
Zalpha has been shown to be sequence nonspecific.
Paul Schimmel
The Skaggs Institute for Chemical Biology,
The Scripps Research Institute,
Beckman Center,
10550 North Torrey Pines Road,Z
La Jolla, CA 92037
The algorithm of the genetic code relates specific amino acids to trinucleotides. The accuracy of the amino-acid-trinucleotide relationship depends significantly on the fine structure discrimination of closely similar amino acids by tRNA synthetases. This discrimination is RNA-dependent. In the discrimination between the closely similar isoleucine and valine side chains by isoleucyl-tRNA synthetase, fine structure differences are only detected in the context of tRNA. Specific nucleotides in the tRNA structure are required for this discrimination. These nucleotides are distinct from those required for aminoacylation. Fine structure recognition occurs at a special site on the enzyme located about 25 angstroms from the active site for aminoacylation. This special site contains the catalytic center for editing, where misactivated aminoacyl linkages are hydrolyzed. Thus, the role of the RNA (i.e., tRNA) is to stimulate translocation of the misactivated amino acid from the active site to the site for editing. The translocation of a misactivated aminoacyl-containing substrate has been detected and the kinetics of RNA-dependent translocation has been investigated in some detail, using a special fluorescence assays.
Bianca Sclavi, Pascal Roux, Malcolm Buckle and Henri Buc
Unité de Physicochimie des Macromolecules Biologiques,
Institut Pasteur,
75015, Paris, France
Bacterial gene transcription in vivo is kinetically regulated at various steps of transcription initiation: promoter recognition and binding by the RNA polymerase, isomerisation to a transcriptionally competent open complex and escape into elongation. The transition between these phases is accompanied by substantial conformational rearrangements of both the protein and the DNA (1). We have applied the method of time-resolved DNaseI footprinting (2) and time-resolved laser crosslinking (3) to measure the rates of formation of key intermediates on the pathway to open complex formation for the E. coli RNA Polymerase on the lac UV5 promoter. These studies on the wild type and mutant DNAs allow us to describe the mechanism by which this polymerase directs the opening of the DNA double helix and the correct positioning of its active site at the initiation start site. In addition the direct measurement of the rates of formation of intermediates will allow us to determine which step on the pathway is the target for different activator and repressor proteins.
References and Footnotes
1. DeHaseth, P., Zupancic, M.L., Record, M.T., Jr., RNA
Polymerase-Promoter Interactions: the Comings and Goings of RNA Polymerase.
Journal of Bacteriology, 180, 3019-3025, 1998.
2. Hsieh, M., Brenowitz, M., Quantitative kinetics footprinting of protein-DNA
association reactions. Methods in Enzymology, 274, 478-92, 1996.
3. Buckle, M., Pemberton, I.K., Jacquet, M.-A., Buc, H., The kinetics of
sigma subunit directed promoter recognition by E. coli RNA polymerase. Journal
of Molecular Biology, 285, 955-964, 1999.
Sepideh Khorasanizadeh, Ramon Campos-Olivas and Michael F. Summers*
Howard Hughes Medical Institute,
University of Maryland Baltimore County,
Baltimore, MD 21250
*Author to whom correspondence should be addressed. Phone: (410) 455-2718;
Fax: (410) 455-1174, E-mail: summers@hhmi.umbc.edu
All retroviruses contain a central capsid core particle that encapsidates the viral RNA genome. The core forms during viral maturation by condensation of approximately 2,000 copies of the capsid protein. The solution structure and dynamics of the capsid protein (CA) from the human T-cell leukemia virus type-I (HTLV-I), a retrovirus that causes T-cell leukemia and HTLV-I-associated myelopathy in humans, is shown to consist of independent N- and C-terminal domains connected by a flexible linker. The domains are structurally similar to the N-terminal "core" and C-terminal "dimerization" domains, respectively, of human immunodeficiency virus type-1 (HIV-1) CA, although several important differences exist. Hydrophobic residues near the major homology region (MHR) are buried in HTLV-I CA, which is monomeric in solution, whereas analogous residues in the HIV-1 CA C-terminal domain promote dimerization. These structural differences appear to be related to, and possibly controlled by, the oxidation state of conserved cysteines, which are reduced in the HTLV-I CA solution structure but form a disulfide in the HIV-1 CA crystal structure. The results are consistent with an oxidative trigger mechanism for retroviral capsid assembly/disassembly.
J. F. Neault, M. Hermann and H. A. Tajmir-Riahi*
Department of Chemistry-Biology,
University of Québec at Trois-Rivières,
C.P. 500, TR (Québec) Canada G9A 5H7
*Author to whom correspondence should be addressed. Fax: 819-376-5084, E-mail:
tajmirri@uqtr.uquebec.ca
Taxol (paclitaxel) (structure 1) is an anticancer drug (1) which interacts with microtubules proteins, in a manner that catalyzes their formation from tubulin and stabilizes the resulting structure (2). However, the taxol-tubulin interaction is affected by the presence of mono-nucleotides and polynucleotides (3). It has also been shown the presence of higher concentration of taxol in the nucleus than in the human lung tumor cell (4). Therefore, this study was designed to examine the interaction of taxol with calf-thymus DNA and yeast RNA in aqueous solution at physiological pH with taxol/polynucleotide(phosphate) molar ratios (r) of 1/80, 1/40, 1/20, 1/10, 1/4 and 1/2. UV-visible and Fourier transform infrared (FTIR) difference spectroscopy were used to characterize the nature of drug-nucleic acids interactions and to establish correlations between spectral changes and the taxol binding mode, binding constant, sequence selectivity, biopolymer secondary structure and structural variations of taxol-polynucleotide complexes in aqueous solution.
Spectroscopic results showed that taxol is an external binder with no affinity towards DNA and RNA intercalation. At low drug concentration r=1/80), no major taxol-polynucleotide interaction occurs, while at higher drug contents (r=1/40, 1/20 and 1/10), there are three major binding sites for taxol on RNA and DNA duplex (a) G-C base pair (at the N-7 guanine), (b) A-T or A-U bases (at O-2 thymine or uridine) and (c) backbone PO2 groups with overall binding constants of K(DNA) =2.5 x 103 M-1 and K(RNA) =7.8 x 103 M-1. The binding constants are similar to those of the other drug-DNA and drug-RNA complexes (5-8). The taxol-DNA interaction is associated with a major reduction of B-DNA structure in favor of A-DNA, while RNA remains in the A-conformation. The drug distributions are about 50% with the G-C bases 30% with the backbone PO2 group and 20% with the A-T or A-U base pairs. At high taxol concentration (1/4 and 1/2), drug aggregation is observed, which does not favor taxol-polynucleotide complexation.
Structure 1
References and Footnotes
1. P. B. Schiff, and S. B. Horwitz, Nature 277,
665-668, 1979.
2. E. Nogales, S. G., Wolf, I. A. Khan, R. F. Luduena, and K. H. Downing,
Nature 375, 424-427, 1995.
3. E. Hamel, A. A. del Campo, M. C. Lowe, and C. M. Lin, J. Biol. Chem.
256, 11887-11894, 1981.
4. M. G. Solis Recendez, F. Bichat, F. Grossin, H. Barbault, D. Khayat,
G. Bastin, Anti-Cancer Treatment 15, 105-110, 1996.
5. J. F. Neault, and H. A. Tajmir-Riahi, J. Biol. Chem. 272, 8901-8904,1997.
6. J. F. Neault, and H. A. Tajmir-Riahi, J. Biol. Chem. 271, 8140-8143,1996.
7. J. F. Neault, and H. A. Tajmir-Riahi, J. Phys. Chem. 102, 1610-1614,1998.
8. J. F. Neault, and H. A. Tajmir-Riahi, Biophys. J. in press, 1999.
Sylvia Eiler, Luc Moulinier, Jean-Claude Thierry and D. Moras
Laboratoire de Biologie Structurale,
Institut de Génétique et de Biologie Moléculaire et
Cellulaire,
CNRS/INSERM/ULP,
1 rue Laurent Fries, BP 163,
67 404 Illkirch Cedex, C.U. de Strasbourg, France
The 2.4 Å crystal structure of the E. coli tRNAAsp-aspartyl-tRNA synthetase (AspRS) complex shows all substrates ready for the transfer of the aspartic acid to the 3'-hydroxyl of the tRNA terminal adenosine. Amino acid residues and, in particular, water molecules involved in the specific recognition of the substrates have been identified. The presence of water molecules vary from one domain to the other and appears to depend on the role of each domain during the recognition process. Two extreme situations are observed concerning the N-terminal and the insertion domain of the E. coli aspartyl tRNA synthetase. The N-terminal domains anchors the anticodon loop of the tRNA through a network of direct hydrophobic and hydrogen bonds closely interweaved. No water molecules are seen at the interface. The situation is different at the interface with the insertion domain, where a network of water mediated interactions is observed. A hybrid situation is observed at the interface between the catalytic domain and the acceptor stem of the tRNA or the hinge domain and the D-stem of the tRNA. In all those situations, the presence of water molecules appears to be associated with the need of larger adaptability and dynamic in the recognition process. On the contrary, any time a specificity of recognition and/or a precise positioning of the substrates is required (for the catalysis for example), the system will tend to avoid as much as possible water molecules as also observed for the recognition of the GCCA single stranded amino acid acceptor end of the tRNA.
Two distinct recognition modes of the tRNAAsp amino acid acceptor stems by their cognate aspartyl-tRNA synthetases in eucaryotes and procaryotes have been observed and will be discussed (1) (2) (3).
References and Footnotes
1. Ruff et al., Science 252, 1682-1689, 1991.
2. Cavarelli et al., EMBO J. 13, 327-337, 1994.
3. Eiler et al., 1999, sumitted for publication.
Youri Timsit*
Institut de Biologie Physico-Chimique, CNRS,|
13, rue Pierre et Marie Curie,
Paris 75005 France
*For author correspondence. Phone: 01 43 25 26 09; Fax: 01 43 29 56 45;
E-mail: timsit@ibpc.fr
The close approach of DNA segments participates in many biological functions including DNA condensation and DNA processing. Previous crystallographic studies have shown that B-DNA self-fitting by mutual groove-backbone interaction produces right-handed DNA crossovers. These structures have opened new perspectives on the role of close DNA-DNA interactions in the architecture and activity the DNA crossovers and Holliday junctions (1). The analysis of the crystal packing of B-DNA decamer duplexes has revealed that symmetric left-handed crossovers can be produced by mutual fitting of DNA grooves at the crossing point (2,3). New sequence patterns contribute to stabilize longitudinal fitting of the sugar-phosphate backbone into the major groove. As found in right-handed DNA crossovers, the close approach of DNA segments greatly influences the DNA conformation in a sequence dependent manner.
The detailed molecular views of DNA crossovers of opposite chirality helps to elucidate the role of symmetry and chirality in the recognition of complex DNA structures by protein dimers or tetramers, such as topoisomerase II and recombinase enzymes. For example, the possible modes of assembly of yeast topoisomerase II with right and left-handed tight DNA crossovers has been investigated, using the crystal coordinates of the docking partners (4,5). Taking into account the rules for building symmetric ternary complexes and the structural constraints imposed by DNA-DNA and protein DNA interactions, this analysis has revealed that two geometric solutions could exist, depending on the chirality of the DNA crossovers. The finding of new DNA binding domains which could interact with the crossovers provides structural supports for each model. The structural similarity of a loop containing a cluster of conserved basic residues pointing into the central hole of topoisomerase II and the second DNA binding site of histone H5 which binds DNA crossover is of particular interest. This work provides structural insights for better understanding the role of chirality and symmetry in topoisomerase II-DNA crossover recognition, suggests testable experiments to further elucidate the structure of ternary complexes and raises new questions about the relationships between the mechanism of strand-passage and strand exchange catalyzed by the enzyme.
References and Footnotes
1. Timsit, Y. and Moras, D., Q. Rev. Biophys. 29,
279-307, 1996.
2. Timsit, Y. and Moras, D., EMBO J. 13, 2737-2746, 1994.
3. Timsit, Y., Shatzky-Schwartz, M., and Shakked, Z., J. Biomolec. Struct.
Dyn. 16, 775-786, 1999.
4. Timsit, Y., Duplantier, B., Jannink, G. and Sikorav, J.L., J. Mol.
Biol. 284, 1289-1299, 1998.
5. Sikorav, J.L., Duplantier, B., Jannink, G. and Timsit, Y., J. Mol.
Biol. 284, 1279-1287, 1998.
Claudine Mayer-Jung1, Dino Moras1 and Youri Timsit2*
1Laboratoire de Biologie Sructurale,
1, rue Laurent Fries, BP 163 67404 Illkirch Cedex France
2 Institut de Biologie Physico-Chimique, CNRS
13, rue Pierre et Marie Curie PARIS 75005 France
*Author to whom correspondence should be addressed. Phone: 01 43 25 26 09;
Fax: 01 43 29 56 45; E-mail: timsit@ibpc.fr
The influence of cytosine methylation on DNA recognition and hydration is analysed. The crystal structures of B-DNA dodecamer and A-DNA decamer duplexes methylated at CpG sites, the target sequence for DNA-methylase, show that the methyl group can promote DNA-DNA recognition and DNA hydration at the modified CpG sequences through the formation of C-H...O interactions. In the dodecamer structure, the two methyl groups form a clamp which traps the incoming phosphate in the groove-backbone interaction (1). This geometry allows the formation of new C-H...O interactions anchoring the methyl groups to the anionic oxygen atoms of the phosphate. This finding relates cytosine methylation to the formation of higher-order DNA structures, and could provide new insights for understanding the mode of action of DNA methylation in genetic inactivation.
In addition, the cristallographic analysis of the hydration pattern around methylated CpG steps in high resolution structures of A-DNA decamers has revealed that the methyl groups of cytosine residues are well hydrated (2). In comparing the native structure with two structuraly distinct forms of the decamer d(CCGCCGGCGG) fully methylated at the its CpG steps, this study has also shown that in certain structural and sequence contexts, the methylated cytosine base can be more hydrated than the unmodifed one. These water molecules seem to be stabilized in front of the methyl group through the formation of C-H...O interactions. These structures provide the first observation of magnesium cations bound to the major groove of A-DNA and reveal two distincts modes of metal binding in methylated and native duplexes. These findings predict that methylated cytosine bases could be recognized by polar residues in protein or DNA, either directly or indirectly, through its tightly bound water molecules.
References and Footnotes
1. Mayer-Jung, C., Moras, D. and Timsit, Y., J. Mol.
Biol. 270, 328-335, 1997.
2. Mayer-Jung, C., Moras, D. and Timsit, Y., EMBO J. 17, 2709-2718,
1998.
Eimer Tuite1*, Per Lincoln1,
Hans-Christian Becker1, Björn Önfelt1, Donats Erts2 and Bengt
Nordén1
1Department of Physical Chemistry,
Chalmers University of Technology,
S-412 96 Göteborg, Sweden
2Institute of Chemical Physics,
University of Latvia,
LV-1586 Riga, Latvia
*Author to whom correspondence should be addressed. E-mail: etuite@phc.chalmers.se
There has been considerable speculation during the past few years about the possibility that DNA can act as a wire and mediate charge transport over long distances via the pi-systems of the basepair stack, perhaps at high speed. The results from different research groups present a disparate picture but as more and more findings are published, certain patterns are beginning to emerge. It is apparent that when nucleobase radical cations or anions are created within a DNA duplex relatively long range charge transfer can occur, and most likely this occurs by a hopping mechanism. This has been shown for cases in which cations and anions are generated by ionizing radiation or by photoinduced electron transfer, and generally concurs with older data on photoconduction of DNA.
A few studies however, have suggested that DNA may have some conduction properties (perhaps semiconductor-like rather than metallic) even when the bases have not been ionized, and it is towards investigating this phenomenon that our efforts have been principally directed. Two approaches are taken, with emphasis on the former:
Previous work from our laboratory on electron transfer reactions of excited state [Ru(phen)2(dppz)]2+ has indicted that DNA in inefficient in mediating charge transfer processes between non-covalently bound ligands and acts mainly as an anionic scaffold, concentrating the cationic reactants and forcing them into close proximity. Cases that apparently contradict this such as highly efficient quenching by [Rh(phi)2(bpy)]3+ (suggesting effective electron transfer distances of >100 Å) are found to be explained by significant Ru2+-Rh3+ binding cooperativity and substantial quenching only for nearest neighbour complexes. Quenching in DNA-bound donor-bridge-acceptor intercalator pairs has also been studied but there have been no indications of greatly enhanced quenching when the compounds are bound to DNA compared to solution.
References and Footnotes
1. B. Nordén, P. Lincoln, B. Åkerman and E.
Tuite, Metal Ions in Biological Systems, 33, 177, 1996.
2. E. Tuite, P. Lincoln and B. Nordén, J. Am. Chem. Soc., 119,
239, 1997.
3. P. Lincoln, E. Tuite and B. Nordén, J. Am. Chem. Soc., 119,
1454, 1997.
4. S.-D. Choi, M.-S. Kim, S. K. Kim, P. Lincoln, E. Tuite and B. Nordén,
Biochemistry, 36, 214, 1997.
5. E. Tuite, Molecular and Supramolecular Photochemistry, 2, 55,
1998.
Eimer Tuite1*, Peter Nielsen2 and Bengt Norden1
1Department of Physical Chemistry,
Chalmers Tekniska Högskola,
S-412 96 Göteborg, Sweden
2Center for Biomolecular Recognition,
Department of Medical Biochemistry and Genetics, Biochemistry Lab B,
The University of Copenhagen, Blegdamsveg 3c,
DK-2200 Copenhagen N, Denmark
*Author to whom correspondence should be addressed. E-mail: etuite@phc.chalmers.se
PNA containing alternating diaminopurine and 2-thiouracil bases cannot form a self-complementary duplex because of steric hindrance between these modified bases. It can however form a very stable duplex with a complementary DNA strand containing alternating adenine and thymine bases, and can invade duplex DNA of this sequence to produce two DNA:PNA duplexes. This double duplex-strand-invasion has been monitored by CD spectroscopy. The stoichiometry is confirmed to be 1:1 and the binding kinetics increase with PNA concentration and temperature, and decrease with ionic strength. Under pseudo-first order conditions, Arrhenius analysis reveals an ionic strength independent activation enthalpy of ca. 70 kJ mol-1 which indicates that, as for normal PNA triplex-strand-invasion, DNA base pair opening is likely the rate-limiting step.
Andrew H.-J. Wang1* Shao-Yu Su1, Yi-Gui Gao1, Howard Robinson1, Bradford S. McCrary2,
Stephen P. Edmondson2 and John W. Shriver2
1Department of Cell & Structural Biology,
University of Illinois at Urbana-Champaign,
Urbana, IL 61801
2Department of Medical Biochemistry,
School of Medicine,
Southern Illinois University,
Carbondale, Illinois 62901
Sac7d and Sso7d are two small (~7 kD), but abundant, chromosomal proteins from the hyperthermophilic Sulfolobus archaeabacteria. These proteins have high thermal, acid and chemical stability. They bind DNA without marked sequence preference and increase the Tm of DNA by ~40°C. Several complexes of Sac7d and Sso7d with DNA octanucleotides were crystallized in different crystal lattices and their crystal structures solved at high resolution. Sac7d/Sso7d binds in the minor groove of DNA and causes a single step sharp kink in DNA (~60°) by the intercalation of the hydrophobic side chains of Val26 and Met29 from the protein. The intercalation sites are different in the two complexes. Observations of this novel DNA binding mode in three independent crystal lattices indicate that it is not a function of crystal packing. The structural motif may have general relevance to understanding the functions of other DNA binding proteins. Our results may be useful in understanding the chromatin organization of the ancient hyperthermophilic Sulfolobus organisms.
David Wemmer
University of California,
Berkeley CA 94720
NMR structural studies of the natural product distamycin lead to identification of a new binding mode with two molecules running antiparallel, side-by-side in the minor groove. This contrasted with the binding of a single molecule in the particularly narrow groove of polyA like sequences which had been observed crystallographically as well as by NMR. The realization that groove width was a critical determinant of binding affinity lead to resurrection of an idea which first came from the groups of Dickerson and Lown - that replacement of pyrrole rings of the ligand with imidazole should lead to G-C specificity by hydrogen bonding to the guanosine amino group. In fact this works very well when done in the context of the side-by-side complex which fills the normal width groove.
We will discuss recent structural studies of complexes with ligands that have evolved from this original design, that have been synthesized in the laboratory of Prof. Peter Dervan at Caltech. Specifically it has been shown that linking the two ligands covalently leads to a dramatic increase in affinity. Structural studies of "hairpin" linked complexes (in which the two ligands are joined head-to-tail) have given a basis for the observed maximum affinity with a three carbon linker. Complexes with shorter linkers have shown that these can bind in fully overlapped side-by-side complexes, with the linker acting as a flex point to allow the ligand to adapt to the curvature of the DNA. This extends the length of the sequence which can effectively be targetted.
It has been noted that distamycin binds with rather high orientational preference, both in single ligand and side-by-side complexes, however the basis for this preference has remained unclear. We have now characterized a series of ligands in DNA complexes which indicate that interactions of the 'tail' portion of the molecule with the groove specify orientation. We have identified complexes in which contact between the tail and groove is lost, and with it orientational specificity disappears. This work also leads to some new ideas about controling some features of binding through modification of the ligand.
Susan A. White* and Valerie Shipilov
Chemistry Department,
Bryn Mawr College,
Bryn Mawr, PA 19010
*Author to whom correspondence should be addressed. Phone: 610-526-5107;
E-mail: swhite@brynmawr.edu
Under conditions where yeast ribosomal protein L30 is present in excess it can bind to its transcript to inhibit splicing or to its mature mRNA to inhibit translation (1). Thus yeast ribosomal protein L30, formerly called L32 (2), is an autoregulatory protein which interacts directly with the L30 RNA. It is the goal of this work to assess the thermodynamic contribution of RNA and protein residues at the complex interface to the overall binding affinity.
The RNA structural motif important for protein binding is a stem-internal loop-stem and the wild type "2 + 5" internal loop is composed entirely of purines. The protein binding region on the RNA was localized by chemical modification and footprinting studies (3) and RNA sequence requirements were studied by conducting a SELEX experiment on the internal loop region (4). The SELEX experiment suggested a high degree of sequence conservation in the internal loop, with some positions absolutely conserved. We report here the results of mutating each of the internal loop nucleotides to a nucleotide not found in the selection experiment. Three mutations, A12C, G56C, and G58A, individually weaken binding by almost a factor of 100 while G11C, G56A, and A59G decrease binding affinity approximately 30-fold. Finally A57G weakens binding several-fold whereas A55G has near wild-type affinity.
In order to assess the importance of individual amino acid side chains to binding affinity, ten protein mutants were made. Only one amino acid substitution, a phenylalanine to an alanine, dramatically reduces binding affinity and near wild-type affinity is restored when tyrosine is introduced at this position. Preliminary circular dichroism studies suggest that this mutant has near native secondary structure and is almost as stable as wild type protein. At least one basic residue, a lysine, appears to be important for binding but most of the other protein mutants tested do not seriously compromise RNA binding.
References and Footnotes
1. Li, B., Vilardell, J., & Warner, J. R., Proc.
Nat. Acad. Sci. USA 93, 1596-1600, 1996.
2. Mager, W. H., Planta, R. J., Ballesta, J.-P. G., Lee, J. C. , Mizuta,
K., Suzuki, K., Warner, J. R., Woolford, Nucleic Acids Research, 25,
4872-4875, 1997.
3. Li, H., Dalal, S., Kohler, J., Vilardell, J., & White, S., J.
Mol. Biol. 250, 447-459, 1995.
4. Li, H., & White, S. A., RNA 3, 245-254, 1997.
V.K. Ganesh* and D. Velmurugan
Department of Crystallography and Biophysics,
University of Madras, Chennai 600 02, India
*Author to whom correspondence should be addressed. Phone: 1-44-2300122;
E-mail : velu@cyberspace.org
Targeting HIV-1 Reverse Transcriptase is one of the ways of developing anti-viral drugs against Human Immunodeficiency Virus - 1 which causes AIDS. The advantage of targeting Reverse Transcriptase (RT) is that the RT is essential in normal functions of host cells. Non-Nucleoside Reverse TranscriptaseInhibitors (NNRTIs) bind at a site located approximately 10 E from the substrate binding site. HIV-1 is generally very flexible and several drug resistant variants emerge upon drug therapy using NNRTIs. TIBO derivatives are one of the potent NNRTIs. Tyr181Cys mutant is common against most of the including 9-chloro TIBO (R82913) (1). It is also shown that 8 -chloro TIBO (R86183) is quite potent in inhibiting HIV-1 strain containing Tyr181Cys mutation (2). The aim of the present work is to perform modeling and energy calculations using Molecular Mechanics (MM) on complexes of 8-Chloro TIBO with wild type and several drug resistant viral variants to understand the structural aspects of inhibition and drug resistance which will be useful in designing/tailoring drugs that could be potent against wild type as well as to a few drug resistant variants. Binding and Interaction energies were calculated and will be presented.
References and Footnotes
1. Vandamme, A.-M., Debyser, Z., Pauwels, R., DeVreese,
K., Goubau, P., Youle, M., Gazzard, B., Stoffels, P.A., Cauwenbergh, C.F.,
Anne, J., Andries, K., Janssen, P.A.J., Desmyter, J. and De Clerq, E., Res.
Human.Retro., 10, 39-46, 1994.
2. Pauwels, R., Andries, K., Debyser, Z., Kukla, M.J., Schols, D., Breslin,
H.J., Woestenborghs, R., Desmyter, J., Janssen, M.A.C., De Clerq, E. and
Janssen, P.A.J., Antimicrob. Agents.Chemother., 38, 2863-2870.
Robert Kaptein
Bijvoet Center for Biomolecular Research,
Utrecht University,
Padualaan 8,
3584 CH Utrecht,
The Netherlands
The primary role of the DNA-binding domains of repressors and eukaryotic transcription factors is to direct the protein to the DNA target site, where it can exert its function. However, often these domains are responsible for other functional aspects as well. For instance, the lac repressor DNA-binding domain (DBD) undergoes a drastic conformational change (helix folding transition) during DNA binding that is necessary for the induction process in the expression of the lac genes. Recently we solved the structure of a dimeric complex of the lac repressor DBD (1-62) and a 22-bp lac operator. This allowed us to define the changes in strcture and dynamics that occur upon complex formation in great detail.
Another example is the glucocorticoid receptor DBD. Here, conformational changes occur upon binding to the hormone response element, that allow the protein to dimerize and interact with other proteins of the transcriptional machinery. We have recently characterized two mutants of the GR-DBD that have already undergone this change in conformation without binding to DNA. Thus, a structural basis for the altered phenotype of these mutants could be provided. A full description of these allosteric transitions that accompany DNA binding can only be obtained from the study of both structure and dynamics of the protein in isolated form as well as in complex with DNA. Ideally these results should then be correlated with kinetic and thermodynamic studies of the same binding process.
Rejadra P. OjhaÝ, Madan M. Dhingra1, Mukti H. Sarma1, Masayuki
Shibata4, Margaret Farrar2,3,
Christopher J. Turner2 and Ramaswamy H. Sarma1*
1Institute of Biomolecular Stereodynamics,
Dept. of Chemistry,
University at Albany, SUNY
1400 Washington Avenue, Albany NY 12222
2Francis Bitter Magnet Laboratory,
Massachusetts Institute of Technology,
170 Albany Street,
Cambridge MA 02139
3Department of Chemistry
Wheaton College,
Norton MA 02766
4Department of Biophysics,
Roswell Park Cancer Institute,
Buffalo, NY 14263
ÝPermanent Address: Department of Physics,
University of Gorakhpur, Gorakhpur, 273009 India.
*Author to whom correspondence should be addressed. Phone: 518-456-9362;
Fax: 518-452-4955; E-mail: rhs07@cnsvax.albany.edu
Although DNA bending plays a crucial role in several biological processes, very little is known experimentally about the relationship between sugar phosphate conformation and sequence directed bending. In this paper, we determine the coupling constants from 2D NMR experiments and define the sugar phosphate backbone geometry in an 11-mer A-tract DNA duplex. Along each chain of the duplex, we determine the sugar pucker, torsional preferences and conformational averaging about the C3'-O3', C4'-C5' and C5'-O5' bonds for each nucleotide. We are able to define the sequence dependent correlated backbone conformational changes and the propagation of local distortions at the backbone upon DNA bending. While the A-tract itself is in B-form, it displays local polymorphism in that the C4'-C5' torsion of the T-strand becomes increasingly trans towards the 5' end of the A-tract, and the minor groove narrows towards the 3' end. In fact, at the 5' end of the A-tract in one of the strands, at the thymine-purine junction, we detect significant populations of noncanonical B-DNA trans conformation at the C4'-C5' exocyclic bond. This can increase the interphosphate distance and lead to local unwinding of the duplex and rolling of the base pair into the major groove. This will create a kink or hinge. At the 3'-end of the A-tract in the purine-thymine step, the duplex is compressed by the presence of a junction between A and B forms of DNA exclusively in one strand, with consequent reduction of the phosphate-phosphate distance. Structural distortions are extremely localized with little or no propagation. It is likely that transcription factor proteins recognize these preexisting deformations in the free DNA itself and mold it into the matrix of the protein. The coupling constant data clearly enable to rule out the original DNA bending model of Crothers, and several of the experimental findings are consistent with the predictions from the Monte Carlo based generalized B-DNA bending formalism of Zhurkin.
EVENING
LECTURE
9:00 pm, Wednesday, June 16, 1999
Structural Biology of the HIV gp120 Envelope Glycoprotein
Wayne A. Hendrickson,
Howard Hughes Medical Institute,
Department of Biochemistry and Molecular Biophysics,
Columbia University, New York, NY 10032
The entry of human immunodeficiency virus (HIV) into cells involves interactions between the viral surface and two cellular receptors. The exterior envelope glycoprotein, gp120, binds first to the CD4 glycoprotein and then to a chemokine receptor on the cell surface. This, in turn, triggers a fusion of the viral and cellular membranes and the initiation of infection. HIV eludes the immune system despite the potential for both gp120 and the intact virus to elicit virus-neutralizing antibodies. We have prepared crystals of an HIV-1 gp120 core complexed with a two-domain fragment (D1D2) of human CD4 and an antigen-binding fragment (Fab) of a neutralizing antibody that blocks chemokine receptor binding, (1), and the structure of this complex has been determined at 2.5Ä resolution, (2). This structure provides a framework for understanding the mechanisms for HIV entry into cells and for viral evasion of immune responses.
References and Footnotes
1. P.D. Kwong, R. Wyatt, E. Desjardins, J. Robinson, J.S.
Culp, B.D. Hellmig, R.W. Sweet, J. Sodroski and W.A. Hendrickson, "Probability
Analysis of Vafriational Crystallization and its Application to gp120, the
Exterior Envelope Glycoprotein of Type 1 Human Immunodeficiency Virus (HIV-1),
J. Biol. Chem. 274, 4115-4123 (1999).
2. P.D. Kwong, R. Wyatt, J. Robinson, R.W. Sweet, J. Sodroski and W.A. Hendrickson,
"Structure of an HIV gp120 Envelope Glycoprotein in Complex with the
CD4 Receptor and a Neutralizing Human Antibody," Nature 393,
648-659 (1998).
Mechanisms of Transcription and Transcription Regulation in Single RNA Polymerase Molecules.
J. Gelles*1, H. Yin2, R. Landick3, M.D. Wang4, M.J. Schnitzer5 and S.M. Block5
1Dept. of Biochemistry,
Brandeis Univ.,
Waltham, MA 02454
2Dept. of Physiology,
Tufts Univ. Med. Sch.,
Boston, MA 02111
3Dept. of Bacteriology,
Univ. of Wisconsin,
Madison, WI 53706
4Dept. of Physics,
Cornell Univ.,
Ithaca, NY 14853
5Dept. of Molecular Biology and Princeton Materials
Inst.,
Princeton Univ.,
Princeton, NJ 08544.
*Author to whom correspondence should be addressed. E-mail: gelles@brandeis.edu
RNA polymerase is simultaneously a molecular motor and a central component of an elaborate signal transduction system. As a molecular motor, it uses the free energy liberated by the polymerization of nucleoside triphosphates to move itself along the DNA template. As a signal transduction protein, the enzyme is the principal target of pathways that regulate gene expression. The mechanisms of these enzyme functions can be difficult to study with conventional biochemical techniques because of the difficulty in synchronizing populations of molecules for kinetic analysis and the inability to directly detect movement in such experiments. These problems can be circumvented with biophysical methods based on light microscopy, which visualize transcription by single, isolated RNA polymerase molecules. This presentation will describe the use of such techniques to study the function of E. coli RNA polymerase, focusing on the mechanism of movement along DNA during transcript elongation and the mechanism of transcription termination at factor-independent terminators
Lester F. Harris*, Michael R. Sullivan and Pamela D. Popken-Harris
David F. Hickok Memorial Cancer Research Laboratory,
Abbott Northwestern Hospital,
800 E. 28th St., Minneapolis, MN 55407
*Author to whom correspondence should be addressed. E-mail:editor@epress.com
We investigated protein/DNA interactions, using molecular dynamics simulations computed between a 10 Angstom water layer model of the 434 cI repressor protein DNA binding domain (DBD) amino acids (R1-69) and of operator (OR1) and its flanks consisting of 28 nucleotide base pairs. Hydrogen bonding interactions were monitored. In addition, van der Waals and electrostatic interaction energies were calculated. Amino acids of the 434 cI repressor DNA recognition helix 3 formed both direct and water mediated hydrogen bonds at cognate codon-anticodon nucleotide base and backbone sites within the OR1 DNA major groove halfsites and flanking regions. In addition, hydrophilic amino acids within the loop between helix 3 and helix 4 have strong electrostatic attraction to codon-anticodon nucleotides located within the central nucleotides of the minor groove between the OR1 DNA major groove halfsites These interactions together induced significant structural changes in the operator DNA manifested by overtwisting of the central nucleotide base pairs and narrowing of the minor groove between the DNA major groove halfsites. Finally, these findings offer a code for site specific DNA recognition by the 434 cI repressor protein.
Juan C. Morales and Eric T. Kool*
Department of Chemistry,
University of Rochester,
Rochester, NY 14627
*Author to whom correspondence should be addressed. E-mail: etk@etk.chem.rochester.edu
Recent x-ray crystal structures of DNA polymerases bound to duplexes
have identified amino acid sidechains hydrogen bonding to minor groove acceptor
atoms(1,2), implicating them as potentially important to insertion and extension
of base pairs during DNA replication. To probe the energetic importance
of these minor groove interactions, as well as the effects of base pair
geometry and Watson-Crick pairing, we have examined the ability of the Klenow
fragment of E. coli DNA Polymerase I to synthesize and extend base pairs
involving nucleoside analogs that lack either Watson-Crick or minor groove
hydrogen bonding groups. A new deoxyadenosine analog, 1 (Q), which has adenine's
minor groove acceptor but not Watson-Crick pairing ability(3), and 2 (Z),
which lacks both Watson-Crick and minor groove hydrogen bonding groups(4,5),
are examined for their effects in both primer and template strands. It is
found that a single minor groove hydrogen bonding acceptor can make up to
a 300-fold difference in efficiency of base pair extension, suggesting that
minor groove interactions play an important role in successful replication.
Minor groove interactions are more important for extension than for insertion
of base pairs with this enzyme, and this effect is stronger in the primer
strand than the template strand. Importantly, the polymerase may be even
more highly sensitive to base pairing geometry in extension of base pairs
than in inserting them in the first place.
References and Footnotes
1. Doublié, S., Tabor, S., Long A. M., Richardson,
C. C., Ellenberger, T., Nature , 391, 251-258, (1998).
2. Kiefer, J. R.; Mao, C.; Braman, J. C.; Beese, L. S., Nature , 391,
304-307, (1998).
3. Morales, J.C., Kool, E. T., J. Am. Chem. Soc., in press, (1999).
4. Guckian, K. M., Morales, J. C., Kool, E. T., J. Org. Chem., 63,
9652-9656, (1998).
5. Morales, J. C., Kool, E. T., Nature Struct. Biol., 5, 950-954,
(1998).
David R. Langley
Bristol Myers Squibb P.R.I.,
Department of Macromolecular Structure,
P.O. Box 5100, Room 458B,
Wallingford, CT 06492
*For author correspondence. Phone: (203) 677-6656; Fax: (203) 677-7702;
E-mail: david@viper.wfd.pri.bms.com or langleyd.bms.com
The BMS Nucleic Acid force field (1) was developed and validated using a series of Molecular Dynamic (MD) Simulations. The result is a force field that closely reproduces the experimentally determined nucleic acid structure for B-DNA and A-RNA in low salt environments and A-DNA in low water activity solutions, such as 4M NaCl or 75% ethanol. Several A -> B-DNA simulations have been carried out in zero added salt solutions. B -> A-DNA simulations have also been carried out on d(GGGCCC)2 in a 75% ethanol environment. These crossover simulations closely converge, less than 1Å RMSD, with simulations starting from the target conformation. Consistent with experimental observations (2,3), the simulation of (AAATTT)2 in a 75% ethanol solution correctly converted from A -> B-DNA.
To further evaluate the force field's ability to reproduce both environment and sequence dependent DNA conformations multi-nanosecond MD simulations were carried out on the d(C5T5).d(A5G5) decamer under 3 different environmental conditions (zero added salt, 4M NaCl and 75% ethanol). This deoxyoligonucleotide contains an A-philic C/G homopolymeric sequence coupled to a B-philic T/A homopolymeric sequence. Sequences of this type have been shown to exist in the B-form under low salt environments (3,4) and form an A/B junction under high salt (4) or water/TFE cond