Bunching Effect in Single-Molecule T4 Lysozyme Nonequilibrium Conformational Dynamics under Enzymatic Reactions

Abstract

The bunching effect, implying that conformational motion times tend to bunch in a finite and narrow time window, is observed and identified to be associated with substrate-enzyme complex formation in T4 lysozyme conformational dynamics under enzymatic reactions. Using single-molecule fluorescence spectroscopy, we have probed T4 lysozyme conformational motions under the hydrolysis reaction of polysaccharide of E. coli B cell walls by monitoring the fluorescence resonant energy transfer (FRET) between a donor-acceptor probe pair tethered to T4 lysozyme domains involving open-close hinge-bending motions. On the basis of the single-molecule spectroscopic results, molecular dynamics simulation, and a random walk model analysis, multiple intermediate states have been estimated in the evolution of T4 lysozyme enzymatic reaction active complex formation (Chen, Y.; Hu, D.; Vorpagel, E. R.; Lu, H. P. Probing single-molecule T4 lysozyme conformational dynamics by intramolecular fluorescence energy transfer. J. Phys. Chem. B 2003, 107, 7947-7956). In this Article, we report progress on the analysis of the reported experimental results, and we have identified the bunching effect of the substrate-enzyme active complex formation time in T4 lysozyme enzymatic reactions. We show that the bunching effect, a dynamic behavior observed for the catalytic hinge-bending conformational motions of T4 lysozyme, is a convoluted outcome of multiple consecutive Poisson rate processes that are defined by protein functional motions under substrate-enzyme interactions; i.e., convoluted multiple Poisson rate processes give rise to the bunching effect in the enzymatic reaction dynamics. We suggest that the bunching effect is likely common in protein conformational dynamics involved in conformation-gated protein functions.

Observing Lysozyme’s Closing and Opening Motions by High-Resolution Single-Molecule Enzymology

“…23–26 The SWNT current I ( t ) for a lysozyme-conjugated nanocircuit is featureless around a steady baseline. Furthermore, lysozyme variants with mutated active site residues known to abrogate enzymatic catalysis also result in a featureless I ( t ).…”

“…Single-molecule distributions of τ closed and τ open followed Poisson statistics and had mean durations that were in excellent agreement with previous FRET studies of T4 lysozyme. 23–26 …”

“…The two- state model does not apply if lysozyme’s motions are limited by more substantial energy barriers; but FRET experiments observe lysozyme accessing a range of conformations, including the closed conformation, at high frequency and without any apparent stabilization when substrate is absent. 26 …”

Probing Single-Molecule Enzyme Active-Site Conformational State Intermittent Coherence

“…18,23 In contrast, along the conformational coordinates, the formation of substrate-enzyme complex and the dissociation of product-enzyme complex are significantly slower on the microseconds to seconds time scales, usually involving large conformational motions that are observable by single-molecule FRET measurements. 18, 23 (2) Enzymatic reaction dynamics in the chemical coordinates shows Markovian or non-Markovian, or even power-law dynamics, 1, 20, 50–63 whereas enzymatic dynamics along the conformational coordinates typically involve with multiple-step conformational motions, such as, bunching 27, 44 and even oscillatory conformational changes 64, 65 (3) The rate-limiting step can be along either conformational or chemical reaction nuclear coordinates. In the measurements of single-molecule conformational dynamics and enzymatic turnovers of HPPK, what we have probed is the dynamics in the conformational coordinate that is essential for forming the active complex for a pyrophosphoryl transfer reaction.…”

The chemical dynamics of nanosensors capable of single-molecule detection

“…For the site probability calculations, we assume a Poisson rate process convolution model described in previous studies. 37,57,58 We consider the probability of a site going from an unquenched to a quenched state as P 1 and the probability of a site going from a quenched to an unquenched state as P 2 . Therefore, the probability of an unquenched site remaining unquenched is 1−P 1 , and the probability of a quenched site remaining quenched is 1−P 2 .…”