Ultrasensitive transient time-resolved monitoring of anisotropic relaxation in NADH with sub-picosecond resolution

Abstract Interconversions between nucleic acid structures play an important role in transcriptional and translational regulation and also in repair and recombination. These interconversions are frequently promoted by nucleic acid chaperone proteins. To monitor their kinetics, Förster resonance energy transfer (FRET) is widely exploited using ensemble fluorescence intensity measurements in pre-steady-state stopped-flow experiments. Such experiments only provide a weighted average of the emission of all species in solution and consume large quantities of materials. Herein, we lift these limitations by combining time-resolved fluorescence (TRF) with droplet microfluidics (DmF). We validate the innovative TRF-DmF approach by investigating the well characterized annealing of the HIV-1 (+)/(–) Primer Binding Sequences (PBS) promoted by a HIV-1 nucleocapsid peptide. Upon rapid mixing of the FRET-labelled (–)PBS with its complementary (+)PBS sequence inside microdroplets, the TRF-DmF set-up enables resolving the time evolution of sub-populations of reacting species and reveals an early intermediate with a ∼50 ps donor fluorescence lifetime never identified so far. TRF-DmF also favorably compares with single molecule experiments, as it offers an accurate control of concentrations with no upper limit, no need to graft one partner on a surface and no photobleaching issues.

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Direct real-time detection of the structural and biochemical events in the myosin power stroke

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.1514859112 ◽

2015 ◽

Vol 112 (46) ◽

pp. 14272-14277 ◽

Cited By ~ 52

Author(s):

Joseph M. Muretta ◽

John A. Rohde ◽

Daniel O. Johnsrud ◽

Sinziana Cornea ◽

David D. Thomas

Keyword(s):

Force Generation ◽

Actin Binding ◽

Power Stroke ◽

Transient Time ◽

Time Resolved ◽

Phosphate Release ◽

Biochemical Kinetics ◽

Temporal Coupling ◽

Muscle Myosin ◽

Kinetics Of

A principal goal of molecular biophysics is to show how protein structural transitions explain physiology. We have developed a strategic tool, transient time-resolved FRET [(TR)2FRET], for this purpose and use it here to measure directly, with millisecond resolution, the structural and biochemical kinetics of muscle myosin and to determine directly how myosin’s power stroke is coupled to the thermodynamic drive for force generation, actin-activated phosphate release, and the weak-to-strong actin-binding transition. We find that actin initiates the power stroke before phosphate dissociation and not after, as many models propose. This result supports a model for muscle contraction in which power output and efficiency are tuned by the distribution of myosin structural states. This technology should have wide application to other systems in which questions about the temporal coupling of allosteric structural and biochemical transitions remain unanswered.

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Heart failure drug changes the mechanoenzymology of the cardiac myosin powerstroke

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.1611698114 ◽

2017 ◽

Vol 114 (10) ◽

pp. E1796-E1804 ◽

Cited By ~ 37

Author(s):

John A. Rohde ◽

David D. Thomas ◽

Joseph M. Muretta

Keyword(s):

Heart Failure ◽

Atp Hydrolysis ◽

Cardiac Contractility ◽

Cardiac Myosin ◽

Transient Time ◽

Time Resolved ◽

Phosphate Release ◽

Omecamtiv Mecarbil ◽

Biochemical State ◽

Kinetics Of

Omecamtiv mecarbil (OM), a putative heart failure therapeutic, increases cardiac contractility. We hypothesize that it does this by changing the structural kinetics of the myosin powerstroke. We tested this directly by performing transient time-resolved FRET on a ventricular cardiac myosin biosensor. Our results demonstrate that OM stabilizes myosin’s prepowerstroke structural state, supporting previous measurements showing that the drug shifts the equilibrium constant for myosin-catalyzed ATP hydrolysis toward the posthydrolysis biochemical state. OM slowed the actin-induced powerstroke, despite a twofold increase in the rate constant for actin-activated phosphate release, the biochemical step in myosin’s ATPase cycle associated with force generation and the conversion of chemical energy into mechanical work. We conclude that OM alters the energetics of cardiac myosin’s mechanical cycle, causing the powerstroke to occur after myosin weakly binds to actin and releases phosphate. We discuss the physiological implications for these changes.

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

Biophysical Journal ◽

10.1016/j.bpj.2009.12.2345 ◽

2010 ◽

Vol 98 (3) ◽

pp. 432a

Author(s):

Roman V. Agafonov ◽

Igor V. Negrashov ◽

Sarah E. Blakely ◽

Margaret A. Titus ◽

Yuri E. Nesmelov ◽

...

Keyword(s):

Structural Dynamics ◽

Transient Time ◽

Time Resolved

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Structural and Biochemical Kinetics of Cardiac Myosin and its Perturbation by a Known Heart Failure Drug Investigated with Transient Time-Resolved FRET

Biophysical Journal ◽

10.1016/j.bpj.2015.11.1598 ◽

2016 ◽

Vol 110 (3) ◽

pp. 296a ◽

Cited By ~ 1

Author(s):

John A. Rohde ◽

David D. Thomas ◽

Joseph M. Muretta

Keyword(s):

Heart Failure ◽

Cardiac Myosin ◽

Transient Time ◽

Time Resolved ◽

Biochemical Kinetics ◽

Kinetics Of

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Picosecond Time-Resolved Amorphization Velocities in Silicon

MRS Proceedings ◽

10.1557/proc-205-99 ◽

1990 ◽

Vol 205 ◽

Author(s):

C.A. Macdonald ◽

B.E. Homan

Keyword(s):

Transition State ◽

Liquid Layer ◽

Thermal Activation ◽

Transition State Theory ◽

Interface Velocity ◽

State Theory ◽

Limited Growth ◽

Layer Depth ◽

Transient Time ◽

Time Resolved

AbstractTransient time-resolved picosecond reflectivity measurements were performed on silicon melted by a 40 ps ultraviolet (308 nm) beam. These were compared to simulations of reflectivity versus amorphous and liquid layer depth and of amorphous interface velocity for various models including transition state theory and density limited growth, both with and without thermal activation.

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