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Dive into the research topics where Igor V. Negrashov is active.

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Featured researches published by Igor V. Negrashov.


Proceedings of the National Academy of Sciences of the United States of America | 2009

Structural dynamics of the myosin relay helix by time-resolved EPR and FRET.

Roman V. Agafonov; Igor V. Negrashov; Yaroslav V. Tkachev; Sarah E. Blakely; Margaret A. Titus; David D. Thomas; Yuri E. Nesmelov

We have used two complementary time-resolved spectroscopic techniques, dipolar electron–electron resonance and fluorescence resonance energy transfer to determine conformational changes in a single structural element of the myosin motor domain, the relay helix, before and after the recovery stroke. Two double-Cys mutants were labeled with optical probes or spin labels, and interprobe distances were determined. Both methods resolved two distinct structural states of myosin, corresponding to straight and bent conformations of the relay helix. The bent state was occupied only upon nucleotide addition, indicating that relay helix, like the entire myosin head, bends in the recovery stroke. However, saturation of myosin with nucleotide, producing a single biochemical state, did not produce a single structural state. Both straight and bent structural states of the relay helix were occupied when either ATP (ADP.BeFx) or ADP.Pi (ADP.AlF4) analogs were bound at the active site. A greater population was found in the bent structural state when the posthydrolysis analog ADP.AlF4 was bound. We conclude that the bending of the relay helix in the recovery stroke does not require ATP hydrolysis but is favored by it. A narrower interprobe distance distribution shows ordering of the relay helix, despite its bending, during the recovery stroke, providing further insight into the dynamics of this energy-transducing structural transition.


Proceedings of the National Academy of Sciences of the United States of America | 2011

Structural kinetics of myosin by transient time-resolved FRET

Yuri E. Nesmelov; Roman V. Agafonov; Igor V. Negrashov; Sarah E. Blakely; Margaret A. Titus; David D. Thomas

For many proteins, especially for molecular motors and other enzymes, the functional mechanisms remain unsolved due to a gap between static structural data and kinetics. We have filled this gap by detecting structure and kinetics simultaneously. This structural kinetics experiment is made possible by a new technique, (TR)2FRET (transient time-resolved FRET), which resolves protein structural states on the submillisecond timescale during the transient phase of a biochemical reaction. (TR)2FRET is accomplished with a fluorescence instrument that uses a pulsed laser and direct waveform recording to acquire an accurate subnanosecond time-resolved fluorescence decay every 0.1 ms after stopped flow. To apply this method to myosin, we labeled the force-generating region site specifically with two probes, mixed rapidly with ATP to initiate the recovery stroke, and measured the interprobe distance by (TR)2FRET with high resolution in both space and time. We found that the relay helix bends during the recovery stroke, most of which occurs before ATP is hydrolyzed, and two structural states (relay helix straight and bent) are resolved in each nucleotide-bound biochemical state. Thus the structural transition of the force-generating region of myosin is only loosely coupled to the ATPase reaction, with conformational selection driving the motor mechanism.


Protein Science | 2013

Metal cation controls myosin and actomyosin kinetics.

Yaroslav V. Tkachev; Jinghua Ge; Igor V. Negrashov; Yuri E. Nesmelov

We have perturbed myosin nucleotide binding site with magnesium‐, manganese‐, or calcium‐nucleotide complexes, using metal cation as a probe to examine the pathways of myosin ATPase in the presence of actin. We have used transient time‐resolved FRET, myosin intrinsic fluorescence, fluorescence of pyrene labeled actin, combined with the steady state myosin ATPase activity measurements of previously characterized D.discoideum myosin construct A639C:K498C. We found that actin activation of myosin ATPase does not depend on metal cation, regardless of the cation‐specific kinetics of nucleotide binding and dissociation. The rate limiting step of myosin ATPase depends on the metal cation. The rate of the recovery stroke and the reverse recovery stroke is directly proportional to the ionic radius of the cation. The rate of nucleotide release from myosin and actomyosin, and ATP binding to actomyosin depends on the cation coordination number.


Biochemistry | 2004

Direct Detection of Phospholamban and Sarcoplasmic Reticulum Ca-ATPase Interaction in Membranes Using Fluorescence Resonance Energy Transfer †

Benjamin Mueller; Christine B. Karim; Igor V. Negrashov; Howard Kutchai; David D. Thomas


Biochemistry | 2004

SERCA Structural Dynamics Induced by ATP and Calcium

Benjamin Mueller; Min Zhao; Igor V. Negrashov; Roberta Bennett; David D. Thomas


Biophysical Journal | 2010

Structural Basis for Uncoupling of Force Generation in the F506A Dictyostelium Myosin Revealed by Time-Resolved EPR and FRET

Roman V. Agafonov; Igor V. Negrashov; Sarah E. Blakely; Margaret A. Titus; Yuri E. Nesmelov; David D. Thomas


Biophysical Journal | 2010

Structural Dynamics by Time-Reolved EPR and Transient Time-Resolved FRET: Application to Myosin

Roman V. Agafonov; Igor V. Negrashov; Sarah E. Blakely; Margaret A. Titus; Yuri E. Nesmelov; David D. Thomas


Biophysical Journal | 2010

Relay Loop Stabilizes the Force-Generating Region in Myosin

Yuri E. Nesmelov; Roman V. Agafonov; Igor V. Negrashov; Sarah E. Blakely; Margaret A. Titus; David D. Thomas


Biophysical Journal | 2010

A Time Resolved Fluorescence Spectrometer with Sub-Millisecond Data Acquisition Time

Joseph M. Muretta; Igor V. Negrashov; David J. Kast; Roman V. Agafonov; Piyali Guhathakurta; Ewa Prochniewicz; Yuri E. Nesmelov; Greg Gillispie; David D. Thomas


Biophysical Journal | 2009

Transient Dynamics of the Force-Generating Domain in Myosin During the Recovery Stroke

Yuri E. Nesmelov; Roman V. Agafonov; Igor V. Negrashov; Sarah E. Blakely; Margaret A. Titus; David D. Thomas

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Yuri E. Nesmelov

University of North Carolina at Charlotte

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