Go to research details of CBB faculty:
Prof. Rabi Ann Musah
Prof. Li Niu
Prof. Ramaswamy H. Sarma
Prof. Charles Scholes
Figure 1. Q-band ENDOR Cavity designed for tuning cavity under pumped helium. The gold-plated cylinder contains the EPR cavity while gear-drives provide adjustment of cavity dimensions and coupling. Detailed schematics available in Rev. Sci. Instrum. 67 1996, 2134
Rhodopsin, Figure 2. ENDOR spectra comparing the 14N-histidine ligand hyperfine structrue as perturbed by nitrite substrate binding.
Figure 3. Dielectric Resonator stopped-flow EPR probe with Wiskind Mixer (Update Instrument, Inc.). Details described in Rev. Sci. Instrum. 65 1994, 68.
Dr. Andrei Veselov, researcher in chemical physics, Ph.D. from Institute of Chemical Kinetics and Combustion, Novosibirsk, Russia. (firstname.lastname@example.org)
Dr. Vladimir Grigoryants, researcher in chemical physics, Ph.D. from Institute of ChemicalKinetics and Combustion, Novosibirsk, Russia. (email@example.com)
Mr. Yiwei Zhao, inorganic chemistry graduate student, BS (chemistry) from Fudan University, PRC. (YZ7171@albany.edu)
Ms. Yuhua Sun, biochemistry graduate student, BS (biology) from Fudan University, PRC. (firstname.lastname@example.org)
Mr. Harold Taylor, electronics technician.
Dr. Andrzej Sienkiewicz, Institute of Physics, Academy of Sciences, Warsaw, Poland.
Charlie Scholes Laboratory
Professor of Chemistry, Ph.D., 1969, Yale , Postdoctoral Fellow: 1969-1970, Oxford; 1970-1973, UCSD,
Ph: 518) 442-4551; fx: 518-452-3462;
EPR and ENDOR of Paramagnetic Biomolecules
The purpose of our work is to understand how biological systems that contain metal centers work. The major techniques are EPR (electron paramagnetic resonance), X and Q-band ENDOR (electron nuclear double resonance). We are exploiting the Q-band (34 GHz) ENDOR that we have recently developed, and we use EPR/ENDOR for monitoring paramagnetic intermediatesr proteins.
The ENDOR Apparatus Development
We have developed a Q-band (34 GHz) ENDOR probe (Figure 1) for continuously tuning the cavity resonant frequency under pumped liquid helium conditions over a broad frequency range (> 2.0 GHz) [Rev. Sci. Instrum. 67 1996, 2134]. Such a range compensates for cooling-induced cavity contraction, presence of cryogenic in the cavity, and insertion of high dielectric frozen aqueous samples. The tuning range is indispensable for use with present commercial Q-band bridges whose low noise Gunn diode oscillators provide a small ~50 MHz tuning range. A similar system patterned after our design is now used by Isaacson at UCSD for semiquinone ENDOR at nitrogen temperatures. For detailed design information contact A. Sienkiewicz (email@example.com). B. Smith (firstname.lastname@example.org)) of our SUNY Albany machine shop routinely manufactures such microwave apparatus for ourselves and other institutions.
This metalloenzyme produces large quantities of nitric oxide (NO) from nitrite as part of the agriculturally and economically significant denitrifying cycle. In collaboration with Prof. Jim Shapleigh (email@example.com), Dept. of Microbiology, Cornell, we study the function and metal-centered details of copper-containing nitrite reductase. We have characterized the enzyme and its mutants by a combination of magnetic resonance, kinetic, and electrochemical techniques available in our lab (Biochemistry 37 1998, 6086). Our Q-band ENDOR shows electronic structural change to the copper liganding environment upon nitrite binding (Figure 2) and intimate electronic perturbation of active sites due to mutation that also alters function (Biochemistry 37 1998, 6095).
Bleomycin is an antitumor antibiotic which damages DNA in the presence of metal ions (most notably Fe2+) and dioxygen. Activated bleomycin is the oxygenated form of bleomycin necessary to induce the free radical by which DNA is broken. By Q-band ENDOR we have probed the activated (17O) oxygen (J. Am. Chem. Soc. 117 1995, 7508). Recent work on bleomycin-DNA complexes resolved the distance-dependent hyperfine interaction of 31P on the backbone of DNA substrate as it is specifically bound to bleomycin (J. Am. Chem. Soc. 120 1998, 1030). This work is in collaboration with Dr. R. Burger (firstname.lastname@example.org), Public Health Research Institute, New York.
Paramagnetic Centers of Cytochrome Oxidase
Cytochrome oxidase is the essential terminal enzyme of aerobic respiration from yeast to man. The work is now aimed at the immediate electronic environs of the binuclear heme-copper catalytic center where >90% of the oxygen (O2+) that we breathe is consumed. We are applying Q-band ENDOR to the nitrogens of heme, to nitrogen ligands of the copper, and to the as yet uncertain exchangeable protons and 17O near both metals. This work, directed at finding electronic structural details of partially reduced intermediates, is beyond the power of protein x-ray crystallography to resolve. We collaborate in this work with Dr. R. Gennis, Dept. of Chemistry, U. of Illinois.
Dielectric Resonator (DR)-Based Stopped Flow EPR
The dielectric resonator provides a 30-fold increase with microliter-sized samples in sensitivity over standard EPR cavities. Integrating the DR to an adjacent rapid-mixer provides for the kinetic detection of paramagnetic species on the millisecond time scale.
Application to Protein Folding
For describing the folding/unfolding of cytochrome c, we combined cysteine-specific spin labeling and stopped-flow EPR. The work described in Biochemistry (36 1997, 2884) is the first application to combine the power of cysteine-directed spin labeling with the millisecond time resolution of stopped-flow. The combination means that we can probe time-development of folding/unfolding and probe immobilization at different amino acid locations not previously probed. Selected amino acids in the protein are being individually mutated to cysteine in collaboration with Prof. Jacquelyn S. Fetrow (email@example.com), The Scripps Research Institute.
Technical Development of Dielectric Resonator-Based EPR
This development has centered on our dielectric resonator-based stopped-flow EPR probe (Rev. Sci. Instrum. 65 1994, 68; J. Magnetic Reson. 124 1997, 87; U. S. Patent No. 5598097). With enhanced sensitivity and mL-sized sample volumes, this EPR probe (Figure 3) renders the stopped-flow EPR insensitive to stopped-flow-induced noise, it is robust and easily assembled, and it incorporates a microwave coupling scheme that provides finesse in tuning and freedom from microphonics. In an international collaboration with Polish physicist-engineer Andrzej Sienkiewicz, we have taken the stopped-flow probe to a manufacturable form and have expanded the utility of the probe with rapid field sweep (J. Magnetic Reson. in press). The probe is now available through Update Instrument, Madison, WI (UPDATEINST@aol.com). A submillisecond micro mixer is in the testing stages. Additional design and technical information on DR applications, such as side access DR for fiber studies, is available from Sienkiewicz (firstname.lastname@example.org) and the SUNY Albany Machine Shop (B. Smith, bgs@.albany.edu). clamping.
Harold Taylor (left), Yuhua Sun, Vladimir Grigoryants, Andrei Veselov, Charles P. Scholes, Yiwei Zhao.
Link to Full List of Publications by Professor Scholes
- A. Veselov, J. P. Osborne, R. B. Gennis, & C. P. Scholes, J. Am. Chem. Soc. 122 (2000) 8712-8716. "Q-Band ENDOR (Electron Nuclear Double Resonance) of the Heme o3 Liganding Environment at the Binuclear Center in Cytochrome bo3 from Escherichia coli".
- V. M. Grigoryants, A. Veselov, & C. P. Scholes, Biophysical J. 78 (2000) 2702-2708. "Variable Velocity Liquid Flow EPR Applied to Submillisecond Protein Folding".
- A. Veselov, J. O. Osborne, R. B. Gennis, & C. P. Scholes, Biochemistry 39 (2000) 3169-3175. "Q-Band ENDOR (Electron Nuclear Double Resonance) Study of the High-Affinity Ubisemiquinone Center in Cytochrome bo3 from Escherichia coli"
- A. Sienkiewicz, M. Jaworski, B. G. Smith, P. G. Fajer, & C. P. Scholes, J. Magnetic Reson. 143 (2000) 144-152. "Dielectric-Resonator-Based Side-Access Probe for Muscle Fiber EPR Study".
- A. Sienkiewicz, A. M. da Costa Ferreira, B. Danner, & C. P. Scholes, J. Magnetic Reson. 136 (1999) 137-142. "Dielectric Resonator-Based Flow and Stopped-Flow EPR with Rapid Field Scanning: A Methodology for Increasing Kinetic Information".
- A. Veselov, K. Olesen, A. Sienkiewicz, J. P. Shapleigh, & C. P. Scholes, Biochemistry 37 (1998) 6095-6105. "Electronic Structural Information from Q-band ENDOR on the Type 1 and Type 2 Copper Liganding Environment in Wild Type and Mutant Forms of Copper-Containing Nitrite Reductase".
- K. Olesen, A. Veselov, Y. Zhao, Y. Wang, B. Danner, C. P. Scholes, & J. P. Shapleigh, Biochemistry 37 (1998) 6086-6094. "Spectroscopic, Kinetic, and Electrochemical Characterization of Heterologously Expressed Wild Type and Mutant Forms of Copper-Containing Nitrite Reductase from Rhodobacter sphaeroides 2.4.3".
- A. Veselov, R. M. Burger, & C. P. Scholes, J. Am. Chem. Soc. 120 (1998) 1030-1033. "Q-band ENDOR (Electron Nuclear Double Resonance) of Ferric-Bleomycin and Activated Bleomycin Complexes with DNA: Fe(III) Hyperfine Interaction with 31P and DNA Induced Perturbations to Bleomycin Structure".
- K. Qu, J. L. Vaughn, A. Sienkiewicz, C. P. Scholes, & J. S. Fetrow, Biochemistry 36 (1997) 2884-2897. "Kinetics and Motional Dynamics of Spin Labeled Yeast Iso-1-Cytochrome c: 1. Stopped-flow EPR as a Probe for Protein Folding/Unfolding of the C-terminal Helix Spin Labeled at Cysteine 102".
- M. Jaworski, A. Sienkiewicz, & C. P. Scholes, J. Magnetic Reson. 124 (1997) 87-96. "Double-stacked Dielectric Ring Resonator for Sensitive EPR Measurements".