Gerlinde Bischoff1*, Robert Bischoff2, Eckhard Birch-Hirschfeld3,
Ulrich Gromann1, Sabine Landau1,
Walter-Vesely Meister1, Sergio de A. Bambirra1, Christian Bohley1 and
Siegried Hoffman1
Martin Luther University Halle-Wittenberg
1Institute of Biochemistry, D-06120 Halle (Saale),
Germany
2Depertment of Oto-, Rhino-, Laryngology, Face
and Neck Surgery, D-06110 Halle (Saale), Germany
3Friedrich Schiller University, Institute of
Virology, D-07745 Jena, Germany
*Author to whom correspondence should be addressed. Phone: ++49 345 5524855;
Fax: ++49 345 5527011. E-mail: bischoff@fu-berlin.de; http://www.chemie.fu-berlin.de/~bischoff
Abstract
The interactions of the drugs 2,7-bis[(diethylamino)-ethoxy]-fluoren-9-one dihydrochloride (Tilorone), 2,7-bis[(dipropylamino)-acetamido]-fluoren-9-one dihydrochloride (FA-2), 2'-(4-hydroxyphenyl)-5-(4-methyl-1-piperazinyl)-2,5'-bi-1H-benzimidazole trihydrochloride (Hoechst 33258), and hematoporphyrin IX derivative (HPD) with synthetic self-complementary DNA (36-b.p.; 5'-biotin-spacer-[d(CGCTATATAGCG)]3-3') were studied by SPR (Surface Plasmon Resonance). Monolayers of biotinylated DNA were immobilized on a streptavidin-dextran-gold triple-layer. Small portions of the drugs (approximately 30 pmol/ml) were injected in continuous flow. The mass corresponded to the amount of the bound molecules. Injections of 50 mM sodium hydroxide pulses separated the DNA double strands, releasing the effector molecules. Subsequent treatments with the effectors gave reproducible results. The maximum interaction between drug and DNA was observed in the case of Tilorone. 41 molecules could bind to the 36-b.p. DNA duplex.
To investigate the microscopic behavior in condensed nucleic acid phases, SFM (Scanning Force Microscopy)-imaging and polarizing microscopic observations of DNA-effector complexes were carried out. Supplementary UV-absorption thermal denaturation curves of DNA with the above-mentioned effectors in dilute solutions were measured. As an additional aid to understand the geometries of DNA-drug interactions, computer simulations were performed and compared with the experimental data.
Franci Johansen and Jens Peter Jacobsen*
Department of Chemistry,
Odense University, Odense M, DK-5230 Denmark
*Author to whom correspondence should be addressed. Phone: +45 6557 2506;
Fax: +45 6615 87 80; E-mail: jpj@chem.ou.dk
Abstract
We have used one and two dimensional 1H NMR spectroscopy to characterize the binding of a homodimeric oxazole yellow dye, 1,1'-(4,4,8,8-tetramethyl-4,8-diaza-undecamethylene)-bis-4-(3-methyl-2,3-dihydro-(benzo-1,3-oxazole)-2-methylidene)-quinolinium tetraiodide (YOYO), to oligonucleotides containing the (5'-CTAG-3')2 and the (5'-CCGG-3')2 binding sites in either different oligonucleotides or in the same oligonucleotide. YOYO bis-intercalates strongly in all the oligonucleotides used and binds preferentially to a (5'-CTAG-3')2 binding site in the oligonucleotide d(CGCTAGCG)2 (1). YOYO also binds preferentially to a (5'-CCGG-3')2 sequence in the oligonucleotide d(CGCCGGCG)2 (2) but slightly less favorably than to the (5'- CTAG-3')2 sequence in 1. The binding of YOYO to the d(CGCTAGCCGGCG): d(CGCCGGCTAGCG) (3) oligonucleotide, containing two preferential binding sites, was also examined. YOYO forms mixtures of 1:1 and 1:2 complexes with oligonucleotide 3 in ratios dependent on the relative amount of YOYO and the oligonucleotides in the sample. The binding of YOYO to the oligonucleotide 3 occur sequence selective in the (5'-CTAG-3')2 site and the (5'- CCGG-3')2 site.
We have also used two dimensional 1H NMR spectroscopy to determine the solution structure of the DNA oligonucleotide d(5'-CGCTAGCG-3')2 complexed with YOYO. The determination of the structure was based on a total relaxation matrix analysis of the NOESY cross peaks intensities. DQF-COSY spectra were used to obtain coupling constants for the deoxyribose ring protons. The coupling constants were transformed into angle estimates. The NOE derived distance and dihedral restraints were applied in restrained molecular dynamics calculations. Twenty final structures each were generated for the YOYO-complex from both A-form and B-form dsDNA starting structures giving a total of 40 final structures. Since many NOE contacts were observed between YOYO and dsDNA the resulting structure has a fairly high resolution and allows determination of local features in the dsDNA structure after YOYO binding. The root-mean-square (rms) deviation of the coordinates for the forty structures of the complex was 0.39Å. The local DNA structure is distorted in the complex. The helix is unwound by 106° and has an overall helical repeat of 13 base pairs caused by the bis-intercalation of YOYO. The polypropylene amine linker chain is located in the minor groove of dsDNA. Even though the YOYO chromophore contains an oxygen atom instead of the larger sulphur atom in the corresponding compound, TOTO, the structures establish that YOYO require more space than TOTO in the intercalation sites. This is probably caused by the more rigid and planar chromophores in YOYO compared to TOTO.
V. Kothekar1*, K. Ratha2
and H.K. Prasad2
1Depaertment of Biophysics and
2Department of Biotechnology,
All India Institute of Medical Sciences,
Ansari Nagar, New Delhi- 110 029, India
*Author to whom correspondence should be addressed. Phone: 091-011-6593215;
Fax: 091-011-6862663; E-mail: kothekar@medinst.emet.in
Abstract
Three dimensional structure of the first ninety two residues of histone like protein from Mycobacterium tuberculosis (Hlpmt) and histone of Clostridium pasteurianum (DBHclopa) are obtained here, on the basis of amino acid sequences of the two proteins, making use of secondary structure prediction programs, sequence search and HOMOLOGY based modeling tools available on Internet. The proteins were docked to a 35 base paired GC rich U bend DNA (U35DNA). Structures of proteins Hlpmt and DBHclopa; U35DNA; and complexes: Hlpmt-U35DNA and DBHclopa-U35DNA were optimized by molecular mechanics (MM) and simulated for 260 pico seconds (ps) in vacuum by molecular dynamics (MD) technique using AMBER 4.0 package with Cornell et al force field. The proteins, when simulated alone, showed compaction. DBHclopa showed larger compaction compared with Hlpmt. U35DNA when simulated alone straightened out and assumed a B-form. In the complexes, Hlpmt showed same order of compaction as in absence of DNA, while DBHclopa showed reduced compaction. In the presence of Hlpmt two ends of helicoidal axis of U35DNA came closer, but slightly out of plane, indicative of its role in overwinding and packaging double stranded DNA. DBHclopa did not give rise to DNA overwinding. The results show architectural role of Hlpmt and DBHclopa in DNA packaging and its sequence dependence.
S.B. Dixit and B. Jayaram*
Department of Chemistry,
Indian Institute of Technology,
Hauz Khas,
New Delhi- 110016, India
*Author to whom correspondence should be addressed. Phone: 91-011-6861977;
Fax: 91-011-6862037; E-mail: bjayaram@chemistry.iitd.emet.in
Abstract
Hydrogen bonds have been accredited with a major role historically, in the formation and stabilization of biomolecular structures. The formation of hydrogen bonds at protein-DNA interfaces in aqueous medium involves not only favorable interactions of the donor and acceptor functional groups but also a loss of interactions between these groups with the solvent water. We have investigated the energetics of about 500 potential hydrogen bonds occuring at protein-DNA interfaces incorporating some recent improvements in biomolecular force fields and solvation treatments. We present here results of our assessment of hydrogen bond contributions to the overall standard free energy of formation of protein-DNA complexes obtained with the generalized Born model and finite difference Poisson-Boltzmann methodology for solvation in conjunction with AMBER force field. Our results support the emerging view on the role of electrostatics in general and that of hydrogen bonds in particular which is that hydrogen bonds do not drive protein-DNA complex formation by virtue of the unfavourable cost of the electrostatics of desolvation. They however, act to stabilize the complex once it is formed.
Miguel Ortiz-Lomardía, Emilio Jiménez-García,
Ivan García-Bassets and Fernando Azorin*
Department de Biologia Moledular i Cellular,
Centre d'Investigació i Desenvolupament,
Jordi Girona Salgado 18-26,
08034 Barcelona, Spain
*Author to whom correspondence should be addressed. Phone: 3493-4006137;
Fax: 3493-2045904; E-mail: fambmc@cid.csic.es
Abstract
In the presence of specific metal-ions (namely zinc but also cadmium, cobalt and manganese), d(GA·TC)n DNA sequences can form non-B-DNA conformations. At low metal-ion concentration they form [GA(GA·TC)] intramolecular triplexes but, upon increasing the metal concentration, the formation of (GA·GA) intramolecular hairpins is detected. In this paper we address the question of the specific effects of zinc on the structure of the d(GA·TC)n sequences. In the presence of zinc, the DMS-reactivity of the (GA·GA) hairpins is strongly reduced suggesting a direct interaction of the metal-ion with the N7-group of the guanines. This effect is specific for antiparallel-stranded d(GA)n homoduplexes. No such strong decrease in DMS-reactivity is observed in B-DNA duplexes or in d(GGA)n and d(GGGA)n homoduplexes. In addition, the thermal stability of antiparallel-stranded d(GA)n homoduplexes increases in the presence of zinc. On the contrary, the melting temperature of similar B-DNA molecules decreases upon increasing the zinc concentration. Altogether, these result indicate that zinc plays an specific role on the stabilization of the (GA·GA) intramolecular hairpins.
Anne-Marie Sapse1 and J. William Lown2*
1City University of New York and Rockefeller University,
New York, N.Y. 10021
2Department of Chemistry,
University of Alberta,
Edmonton, AB, Canada T6G 2G2
*Author to whom correspondence should be adrressed. Phone: 403-492-3254;
Fax: 403-492-8231
Abstract
Ab initio calculations (Hartree-Fock) using the 3-21G and the STO-3G Gaussian basis sets were performed on synthetic analogues of the minor groove binding bis-benzimidazole Hoechst 33258 designed to be subject to bioreductive activation. Such compounds have been shown experimentally to react with DNA to exhibit sequence dependent inhibition of human placental helicase and display significant anticancer properties. Geometry optimized conformations and energies were derived. The binding of the optimized conformations of the drugs to both alternating and non-alternating (AT)n and to (G)n·(C)n sequences were studied. The energetics of reaction at alternative DNA base sites are calculated and compared.
Thomas E. Cheatham, III1*, Jayashree Srinivasan2,4, David A. Case2 and Peter
A. Kollman3*
1Laboratory of Biophysical Chemistry,
National Heart, Lung and Blood Institute, 12A-2041,
National Institutes of Health,
9000 Rockville Pike,
Bethesda, MD 20892-5626
2Department of Molecular Biology,
The Scripps Research Institute,
La Jolla, CA 92037
3Department of Pharmaceutical Chemistry,
University of California,
San Francisco, CA 94143-0446
4Current address:
CombiChem, Inc.,
9050 Camino Sante Fe,
San Diego, CA 92121
*Correspondence may be addressed to either author.
Peter A. Kollman- Phone: 415-476-4637; Fax: 415-476-0688; E-mail: pak@cgl.ucsf.edu
Thomas E. Cheatham- Phone: 301-402-0617; Fax: 301-496-2172; E-mail: cheatham@helix.nih.gov
Abstract
Molecular dynamics simulation in explicit solvent and continuum solvent models are applied to investigate the relative stability of A- and B-form helices for two DNA sequences, dA10-dT10 and dG10-dC10 in three structural forms. One structural form is based on an unrestrained molecular dynamics (MD) trajectory starting from a canonical B-DNA structure, the second is based on a MD trajectory starting in a canonical B-DNA structure with the sugars constrained to be C2'-endo and the third simulation started from a canonical A-DNA structure with the sugars constrained to C3'-endo puckers. For the energetic analysis, structures were taken as snapshots from nanosecond length molecular dynamics simulations computed in a consistent fashion in explicit solvent, applying the particle mesh Ewald method and the Cornell et al. force field. The electrostatic contributions to solvation free energies are computed using both a finite-difference Poisson-Boltzmann model and a pairwise Generalized Born model. The non-electrostatic contributions to the solvation free energies are estimated with a solvent accessible surface area dependent term. To estimate the gas phase component of the relative free energy between the various structures, the mean solute internal energies (determined with the Cornell et al. molecular mechanics potential including all pairwise interactions within the solute) and estimates of the solute entropy (using a harmonic approximation) were used. Consistent with experiment, the polyG-polyC (GC) structures are found to be much more A-phillic than the polyA-polyT (AT) structures, the latter being quite A-phobic. The dominant energy components responsible for this difference comes from the internal and van der Waal energies. A perhaps less appreciated difference between the GC and AT rich sequences is suggested by the calculated salt dependence which demonstrates a significantly enhanced ability to drive GC rich sequences towards an A-form structure compared to AT rich sequences. In addition to being A-phobic, the AT structure also has a noticably larger helical repeat than GC and other mixed sequence duplexes, consistent with experiment. Analysis of the average solvent density from the trajectories shows hydration patterns in qualitative agreement with experiment and previous theoretical treatments.
G. Fabriciova, P. Miskovsky*, D. Jancura and V. Lisy
Department of Biophysics, P.J. Safarik University,
Jesenna 5, 04 154 Kosice, Slovakia
*Author to whom correspondence should be addressed. Phone: +421956222986;
Fax: +421956222124; E-mail: misko@kosice.upjs.sk
Abstract
Poly(dI-dC) in H2O and D2O
solution can undergo different equilibrium geometries which strongly depend
on the salt nature and concentration. These structures were studied by classical
Raman spectroscopy in order to monitor a hydrogen-deuterium exchange kinetics
in 8-CH group in inosine. Spectral and isotopic exchange rate changes depending
on NaCl concentration were observed and interpreted on the basis of previously
obtained results from resonance and classical Raman spectroscopy studies
of poly(dI-dC) and hydrogen-deuterium exchange measurements of different
conformations of nucleic acids. It is shown that: i) the Raman spectrum
of low-salt poly(dI-dC) corresponds to the right-handed polymer with characteristic
bands for B conformation, but the value of the retardation factor of isotopic
exchange suggests that this form is not a pure canonical B form and that
it contains some portion of the A form, ii) the Raman spectrum of the high-salt
poly(dI-dC) corresponds to the right-handed polymer with characteristic
bands for both the A and B conformations, iii) the retardation factor of
hydrogen deuterium exchange for the high-salt form of poly(dI-dC) is essentially
higher than in the low-salt form which indicates a dominant presence of
the A form in the high-salt conformation of poly(dI-dC). This leads to the
conclusion that the high-salt conformation of poly(dI-dC) is a mixture of
A and B forms with the predominant A form.
Alexander V. Teplukhin1, George G. Malenkov2 and Valery I. Poltev3*
1Institute of Mathematical Problems of Biology,
Russian Academy of Sciences,
Pushchino, Moscow region, 142292, Russia
2Institute of Physical Chemistry,
Russian Academy of Sciences,
31 Leninsky prospekt,
Moscow, 117915, Russia
3Institute of Theoretical and Experimental Biophysics,
Russian Academy of Sciences,
Pushchino, Moscow region, 142292, Russia
*Author to whom correspondence should be addressed. Phone: +7 096-773-94-30;
Fax: +7 096-779-05-53; E-mail: poltev@venus.ireb.sezpukhov.su
Abstract
The set of atom-atom potential functions specially adjusted to simulation of nucleic acid fragment hydration (Poltev, Grokhlina & Malenkov, J. Biomol. Struct. Dyn. 2, 413, 1984) is extended by including alkaline cation interactions. The choice of new potential functions was realized using experimental data on crystal hydrates of nucleotides and related compounds as well as thermodynamic data on ion solutions. The extended set of potential functions allowes to reproduce many features of interactions between alkaline cations and nucleic acid fragments in water solutions. The sites of preferencial cation localization near bases and phosphate groups were obtained and examined. The potential functions reproduce the dissociation tendency of cation-phosphate group and cation-base complexes in aqueous medium. Pathways of cation dissociations from nucleic acid components have been studied, and metastable water-bridged positions of cations near bases and phosphate group have been revealed.
G. Roxström, I. Velázquez, M. Paulino#
and O. Tapia*
Department of Physical Chemistry,
Uppsala University,
Box 532,
S-85121 Uppsala, Sweden
#Permanent address:
Department of Quantum Chemistry,
Facultad de Química,
Universidad de la República,
11800 Montevideo, Uruguay
*Author to whom correspondence should be addressed. Phone: +46 18 18 25
00; Fax: 018 50 85 42; E-mail: Orlando.Tapia@fki.uu.se
Abstract
Molecular dynamics simulations of the zinc finger domain of protein Zif268, in a complex with a high affinity DNA sequence, yields a globally stable system with small yet significant readjustments with persistence time of the order of 1.1ns. The results confirm the quality of the standard GROMOS87 force field with a corrected solvent-to-solute interaction that does not affect the water-water SPC interactions nor the intra-molecular cohesive forces. Specificity determinants are discussed. The simulations of DNA alone, with the same force field, showed the important role played by the solvent and the symmetry of the counterion distribution. (Tapia & Velázquez, J. Am. Chem. Soc., 119, 5934, 1997) In the present work, this feature was retained when appropriate. The results for root mean square deviations and temperature B-factors illustrate the reliability of this approach. The structure of DNA is held by its interactions with the zinc finger protein. This behavior is not much affected by the slow whithering away of finger-1 from DNA. The factors contributing to the molecular stability found in GROMOS' potential energy function appear to be sufficient to yield stable fluctuation patterns when surrounding medium effects are properly included.
B.J. Premraj and N. Yathindra*
Department of Crystallography and Biophysics,
University of Madras, Guindy CXampus,
Chennai 600 025, India
*Author to whom correspondence should be addressed. Phone: 91-44-235-1367;
Fax: 91-44-235-2594; E-mail: crystal@giasmd01.vsnl.net.in
Abstract
Shape and dimension of the preferred nucleotide repeats in nucleic acids are found to depend on whether the sugar-phosphate linkage is of 2',5' or 3',5' type. It is shown that a nucleotide which is "compact" in 3',5' nucleic acids is rendered "extended" and vice versa for a given sugar pucker. It is interesting that this feature is accompanied by a switch in the preferred sugar ring conformation in 3',5' and 2',5' nucleic acids. 3' ribose and 3' deoxyribose rings (in 2',5' linkages) tend to favour C2' endo and C3' endo puckers respectively in contrast to C3' endo and C2' endo puckers favored by 2' ribose and 2' deoxyribose sugars (in 3',5' linkages).The distinguishable features between the nucleotide repeats of 3',5' and 2',5' nucleic acids need to be recognised while discussing their structural properties, as well as those of a variety of complexes that could be formed involving 2',5' and 3',5' strands of DNA and RNA. Ability and stability, or lack of them, for formation of a specific combination of these complexes may be directly related to the stereochemical constraints imposed as a consequence of conformationally homogeneous or heterogeneous nature of the repeating nucleotides of the complexing chains. As a first step towards delineating stereochemical features that distinguish 2',5' nucleic acids from their naturally occurring isomer A and B type helices have been modelled using the new concept of "compact" and "extended" nucleotide repeat that seemingly unifies helix generation of both types of linkages. Helical models for 2',5' RNA with "dinucleotide" repeat based on the crystal structure of 2',5' ApU have also been obtained.
Kenneth A. Marx1*, Iman Q. Assil1, J.W. Bizzaro1 and R.D.
Blake2Ý
1Department of Chemistry,
University of Massachusetts,
Lowell, MA 01854
2Department of Biochemistry,
Microbiology and Molecular Biology,
University of Maine,
Orono, ME 04469
Ýpresent address:
Williams College,
Williamstown, MA 01267
*Author to whom correspondence should be addressed. Phone: 978-934-3658;
Fax: 978-934-3013; E-mail: Kenneth_Marx@uml.edu
Abstract
The slime mold, Dictyostelium discoideum, possesses an (A+T) rich eukaryotic genome that is being sequenced in the Human Genome Project. High resolution melting curves of isolated total and fractionated nuclear D. discoideum DNA(AX3 strain) were determined experimentally and are compared to melting curves calculated from GENBANK sequences (1.59% of genome) by the statistical thermodynamics program MELTSIM (1), parameterized for long DNA sequences (2,3). The lower and upper temperature limits of calculated melting agree well with the observed melting of total DNA. The experimental curve is unusual in that it contains a number of sharp peaks. MELTSIM allowed us to calculate positional denaturation maps of D. discoideum GENBANK sequence documents containing the 26S, 5.8S and 17S rDNA gene sequences, a major satellite DNA and repetitive sequence family present in 100-200 copies/nucleus. These denaturation maps contain subtransitions that correspond with a number of the experimentally observed peaks, some of which we show to correspond with rDNA gene enriched CsCl gradient fractions of D. discoideum DNA. MELTSIM calculated curves of coding, intron and flanking sequences indicate that both intron and flanking sequences are extremely (A+T) rich and account for most of the low temperature melting. There is no temperature overlap between thermal stabilities of these sequence domains and those of coding DNA. The latter must satisfy triplet codon constraints of higher (G+C) content. These large stability property differences enable a denaturation mapping feature of MELTSIM to clearly distinguish exon positions from those of introns and flanking DNA in long D. discoideum gene containing sequences.
Hanspeter Herzel1*, Olaf Weiss1 and Edward N. Trifonov2
1Institute for Theoretical Biology,
Humboldt University,
Invalidenstr. 43,
10115 Berlin, Germany
2Department of Structural Biology,
Weizmann Institute of Science,
Rehovot 76100, Israel
*Author to whom correspondence should be addressed. Phone: +49 30 2093;
Fax: +49 30 2093-8801; E-mail: h.herzel@biologie.huberlin.de
Abstract
The topological state of genomic DNA is of importance for its replication, recombination and transcription. The wrapping of the DNA around nucleosomes is associated with sequence periodicities (Trifonov and Sussman, Proc. Natl. Acad. Sci. USA, 77, pp. 3816-20). Recently, also the negative supercoiling of eubacterial DNA was related to 11 base pair (bp) periodicity (Herzel et al. Physica A, 249, pp. 449-59). Archaeal plasmids and a virus-like particle from Sulfolobus are positively supercoiled, but the superhelical conformation of archaeal genomic DNA is still uncertain. The problem of superhelicity can now be addressed via a comparative statistical analysis of the available complete genomes. For this purpose one has to look for periodicities which are in phase with the helical repeat of 10-11 bp. Similar periodicities are induced, however, by the amphipatic character of alpha-helices of encoded proteins (Zhurkin, Nucl. Acids Res., 9, pp. 1963-71).
We show that these proteininduced periodicities are extended over a few periods only. The periods of additional longranging oscillations deviate significantly from the value for free DNA. A period of 11 bp in Eubacteria reflects negative supercoiling, whereas the significantly different period of thermophilic Archaea close to 10 bp suggests positive supercoiling of archaeal genomes.
Gita Subba Rao*, Sarika Kataria and Mohd. Imran Siddiqui
Department of Biophysics,
All India Institute of Medical Sciences,
New Delhi- 110029, India
ÝPatent applied for.
*Author to whom correspondence should be addressed. Phone: 91-11-659 4816;
Fax: 91-11-686 2663; E-mail: gitaro@medinst.emet.in
Abstract
HIV- I reverse transcriptase (RT) is a key enzyme involved in the replication of the virus and is a potential target for therapeutic intervention following infection. Several drugs that inhibit the enzyme from acting have been discovered. These include nucleoside-analogue inhibitors such as AZT (zidovudine), ddI and ddC, and non-nucleoside inhibitors such as nevirapine and delavirdine. All of them, however have been found to be of limited clinical utility because the RT becomes rapidly resistant to them on account of point mutations in the enzyme. One way to partly overcome this limitation is to design an inhibitor that has interactions mainly with the backbone and the conserved residues of RT. Using a rational drug-design approach based on the high resolution X-ray crystal structure of the RT-nevirapine complex (1), and the specific design principles of peptides containing dehydro-Alanine (DeltaAla) generated by our theoretical calculations, we present here the design of a peptide inhibitor of RT. Energy minimization and molecular modeling of the interaction of the designed pentapeptide with the nevirapine-binding site indicate that the inhibitor has 60% of its interactions with the conserved regions of RT as compared to 30% in the case of nevirapine, thus making it much less sensitive to mutations in the enzyme.
Lynne Reed Murphy*, Naiqi Li, Jean Baum and Ronald M. Levy
Department of Chemistry,
Rutgers University,
Piscataway, NJ 08854-8087
*Author to whom correspondence should be addressed. Phone: 732-445-3278;
Fax: 732-445-5958; E-mail: lreed@lutece.rutgers.edu
Abstract
Molecular dynamics simulations of alpha-lactalbumin were performed under conditions of neutral pH and low pH in order to study the acid-induced molten globule state. Through the use of experimental techniques such as NMR and CD spectroscopy, molten globules have been characterized as being compact intermediates with secondary structure similar to that of the native protein but with tertiary structure that is disordered. The detailed structure of the molten globule state is unknown, however. Through the use of computer simulations we can study the structural changes which occur upon lowering pH. The simulations presented here differ from previous unfolding simulations in two important ways: the electrostatic interactions are treated more accurately than ever before, and artificially high temperatures are not used to force the protein to unfold. Simulations of 880 psec each were run at pH 7 (control simulation) and pH 2. We concentrate on the interesting changes in the tertiary interactions within the protein with lowering of pH. In particular, there is a loss of native tertiary contacts in the beta domain and interdomain region, and a large decrease in interdomain hydrogen bonds.
Rachel Z. Kramer and Helen M. Berman*
Department of Chemistry,
Rutgers University,
Piscataway, NJ 08854
*Author to whom correspondence should be addressed. Phone: 732-445-4667;
Fax: 732-445-4320; E-mail: berman@adenine.rutgers.edu
Abstract
Since the initial observation by Franklin and Gosling which demonstrated that the relative humidity of the sample affects the conformation of DNA, there have been numerous studies of DNA hydration which have attempted to bridge the gap between the macroscopic behavior of DNA and its microscopic characteristics. Several crystal structures have aided these types of analyses. The complex of dCpG and proflavine with its striking pentagonal arrays of water molecules provided a benchmark for a large number of theoretical analyses. The determination of the crystal structure of more than a full turn of DNA that contained within it a "spine of hydration" led to more studies of oligonucleotides in solution, many theoretical studies, as well as systematic studies of the entire corpus of oligonucleotide crystal structures. Among the lessons learned from these studies was that water is indeed an integral part of DNA structure and that the geometry of the first shell is predictable. Through the theoretical studies of the Beveridge group, it was also shown that some of the "water" behavior might in fact be attributable to ions such as sodium. These predictions for dodecamer structures were validated by high-resolution crystal structure analyses.
Jeffrey Skolnick1*, Andrzej Kolinski1,2 and Angel R. Ortiz1
1Department of Molecular Biology,
The Scripps Research Institute,
10550 N. Torrey Pines Rd.,
La Jolla, CA. 92037 USA
2Department of Chemistry
University of Warsaw
02-093 Warsaw, Poland
*Author to whom correspondence should be addressed. Phone: 1-619-784-8821;
Fax: 1-619-784-8895; E-mail: skolnick@scripps.edu
Abstract
One of the most important unsolved problems of computational biology is prediction of the three-dimensional structure of a protein from its amino acid sequence. In practice, the solution to the protein folding problem demands that two interrelated problems be simultaneously addressed. Potentials that recognize the native state from the myriad of misfolded conformations are required, and the multiple minima conformational search problem must be solved. A means of partly surmounting both problems is to use reduced protein models and knowledge-based potentials. Such models have been employed to elucidate a number of general features of protein folding, including the nature of the energy landscape, the factors responsible for the uniqueness of the native state and the origin of the two-state thermodynamic behavior of globular proteins. Reduced models have also been used to predict protein tertiary and quaternary structure. When combined with a limited amount of experimental information about secondary and tertiary structure, molecules of substantial complexity can be assembled. If predicted secondary structure and tertiary restraints are employed, low resolution models of single domain proteins can be successfully predicted. Thus, simplified protein models have played an important role in furthering the understanding of the physical properties of proteins.
Karen E.S. Tang1,2Ý and Ken A.
Dill2*
1Graduate Group in Biophysics,
2Department of Pharmaceutical Chemistry,
University of California,
San Francisco, CA 94143-1204
ÝCurrent address:
Department of Biochemistry,
University of Minnesota,
St. Paul, MN 55108 -1022
*Author to whom correspondence should be addressed. Phone: 415-476-9964;
Fax: 415-476-1508; E-mail: dell@maxwell.ucsf.edu
Abstract
We study the fluctuations of native proteins by exact enumeration using the HP lattice model. The model fluctuations increase with temperature. We observe a low-temperature point, below which large fluctuations are frozen out. This prediction is consistent with the observation by Tilton et al. [R. F. Tilton, Jr., J. C. Dewan, and G. A. Petsko, Biochemistry 31, 2469 (1992)], that the thermal motions of ribonuclease A increase sharply above about 200K. We also explore protein "flexibility" as defined by Debye-Waller-like factors and solvent accessibilities of core residues to hydrogen exchange. We find that proteins having greater stability tend to have fewer large fluctuations, and hence lower flexibilities. If flexibility is necessary for enzyme catalysis, this could explain why proteins from thermophilic organisms, which are exceptionally stable, may be catalytically inactive at normal temperatures.
Gabriel Castro, Charles A. Boswell and Scott H. Northrup*
Department of Chemistry,
Tennessee Technological University,
Cookeville, TN 38505
*Author to whom correspondence should be addressed. Phone: 931-372-3421;
Fax: 931-372-3434; E-mail: snorthrup@tntech.edu
Abstract
The dynamics of the docking step in the electron transfer reaction between yeast cytochrome c peroxidase and iso-1-cytochrome c has been studied using the Brownian dynamics method. In particular we have calculated the bimolecular rate constant at which a specific complex, the xray crystalline complex, can form in solution by translational and rotational diffusion in a field of force. Complexation criteria have been assessed based on the simultaneous alignment of three atom-atom contacts, as well as alternative criteria. The proteins are able to align one or two contacts at remarkably high rates, in fact, at rates approaching the diffusion-controlled limit for two spheres reactive over their entire surfaces. Three contacts may align, and hence the specific complex may dock, at rates on the order of 108 M-1s-1, which is quite representative of the experimental association rate constant for ET-competent complex(es). The formation of the specific complex is strongly influenced by the favorable electrostatic interaction between these proteins. It is striking that a specific protein-protein complex can form within one order of magnitude as fast as two spherical proteins can touch at any orientation. It remains plausible that the high ET tunneling rate in this system can take place through a single highly favorable specific complex using a single high efficiency pathway. Still the contribution from a nonspecific set of complexes is not ruled out, particularly considering the marginal reproduction of the ionic strength dependence in the formation of the xray complex.
Chandralal M. Hewage, Lu Jiang, John A. Parkinson, Robert Ramage and
Ian H. Sadler*
Department of Chemistry.
University of Edinburgh,
West Mains Road,
Edinburgh EH9 3JJ, UK
*Author to whom correspondence should be addressed. Phone: +44-131-650-4822;
Fax: +44-131-650-4743; E-mail: i.h.sadler@ed.ac.uk
Abstract
The solution structure of a synthetic ETB selective agonist, ET-1[Cys(Acm)1,15, Ala3, Leu7, dAsp8, Aib11] has been solved by 1H NMR and molecular modelling studies. Such solution structures of linear modified peptides in aqueous methanol are being used in an ongoing program of research designed to assist in an understanding of the basic structural requirements for the biological activity of vasoconstrictors. The resulting structure of this peptide is characterised by an alpha-helical conformation between residues Leu6-His16 and by N- and C-termini which assume no defined conformation. A knowledge of the solution structures of this and related peptides, which are ETB selective agonists, are proving to be important in the understanding of how they interact with the ETB receptor.
Emilia M. Pedone1, Simonetta Bartolucci1, Mosè Rossi1 and
Michele Saviano2*
1Dipartimento di Chimica
Organica e Biologica,
Universitá "Federico II" di Napoli,
via Mezzocannone, 16
80134, Napoli, Italy
2Centro di Studio di
Biocristallografia del CNR,
Dipartimento di Chimica,
via Mezzocannone, 4
80134, Napoli, Italy
*Author to whom correspondence should be addressed. Phone: 39-81-5476582;
Fax: 39-81-5527771; E-mail: saviano@chemna.dichi.unina.it
Abstract
The knowledge of the relationship between the three-dimensional structure of a protein and its biological and stability is one of the most challenging problem in protein chemistry, since offers the possibility of changing both the specific action of a protein and its stability.
In this work, we have approached the problem with studies on a protein family, the thioredoxins, using homology procedures, molecular dynamics simulations in vacuo at 300 K and 500 K and in water solution at 300 K, to determine the relationship between the three-dimensional structure of these proteins and their thermal stability. A comparative analysis, using computational approach, was performed between two thioredoxins with different resistance to temperature. Results obtained using the molecular dynamics techniques and minimization procedures give explanations of the experimental data, underlining that these techniques are able to correlate the increase in protein stabilization with the conformational and structural changes caused by single amino acid replacement. In addition, we report the factors that can be used as a guide in protein engineering and in site-directed mutagenesis to increase or decrease thermal stabilization for this protein family.
Harold A. Scheraga*
Baker Laboratory of Chemistry and Chemical Biology,
Cornell University,
Ithaca, NY 14853-1301
*For author correspondence. Phone: 607-255-4034; Fax: 607-254-4700; E-mail:
has5@cornell.edu
Abstract
An earlier theoretical approach to the hydrophobic interaction, based on statistical mechanical treatments of models for liquid water and for aqueous solutions of hydrocarbons, is summarized here. Experimental verification of the theoretical thermodynamic parameters for hydrophobic interactions, and applications of the theory to some aspects of protein structure, are presented.
Gerald S. Manning* and Jolly Ray
Department of Chemistry,
Rutgers University,
610 Taylor Road,
Piscataway, NJ 08854-8087
*Author to whom correspondence should be addressed. Phone: 732-445-2609;
Fax: 732-445-5312; E-mail: gmanning@rutchem.rutgers.edu
Abstract
We review some of the characteristic properties of the structure of polyelectrolyte solutions: the condensed layer of counterions that forms abruptly at a critical threshold charge density on the polymer chain; the more diffuse Debye-Hückel cloud, which is spatially distinct from the condensed layer; and the entropic release of counterions from the condensed layer as a driving force for the binding of oppositely charged ligands. We present a reminder of the basis of our current understanding in a variety of experiments, simulations, and theories; and we attempt as well to clarify some misunderstandings. We present a new analysis of a lattice model that suggests why the limiting laws for polyelectrolyte thermodynamics have proved to be accurate despite the neglect of polymer-polymer interactions in their original derivation. We sketch recent progress in constructing a potential between counterion and polyion. A counterion located in the interface between condensed layer and Debye cloud is repelled from the polyion, creating a sharp boundary between the two counterion populations.
V. Lisy1*, T.Yu. Tchesskaya2
and A.V. Zatovsky2
1Department of Biophysics,
P.J. Safarik University,
Jesenna 5,
041 54 Kosice, Slovakia
2Department of Theoretical Physics,
Odessa State University,
Petra Velikogo 2,
270 100 Odessa, Ukraine
*Author to whom correspondence should be addressed. Phone: +95 62 22986;
Fax: +95 62 22124; E-mail: lisy@kosice.upjs.sk
Abstract
The spectra of Rayleigh scattering of Mössbauer radiation (RSMR) and Mössbauer absorption by globular macromolecules are calculated. The dependence of the spectra parameters on hydration is modeled with the account for thermal low-frequency vibrations of the particles constituting the globule. Deformational motions of the macromolecule fragments leading to deviations from its equilibrium spherical shape are considered introducing collective dynamical variables governed by Langevin equations with random sources of external forces. The macromolecule is modeled by a double-layered sphere: a rigid (elastic) core is surrounded by a porous hydration shell filled with fluid. The dynamical properties of the bound water inside the shell are described by the Debye-Brinkman equations. The degree of hydration is introduced by means of a combination of the mass coefficients of the porous shell with fluid and the mass coefficients in the limiting cases when the flow inside the shell is "frozen" and in the case of free flow. The hydration-dependent Lamb-Mössbauer factor and the elastic fraction of the RSMR are calculated and compared with experimental data from the literature.