Single-Molecule Protein Interaction Conformational Dynamics

Abstract

Protein conformational fluctuations and dynamics, often associated with static and dynamic inhomogeneities, play a crucial role in biomolecular functions. It is extremely difficult to characterize such spatially and temporally inhomogeneous dynamics in an ensemble-averaged measurement, especially when the proteins involve in a multiple-step and multiple-conformation complex chemical interactions and transformations, such as in protein-protein interactions and protein-DNA interactions. Single-molecule spectroscopy is a powerful approach to analyze protein conformational dynamics under physiological conditions, providing dynamic perspectives on a molecular-level understanding of protein structure-function mechanisms.

Single-molecule approaches to characterizing kinetics of biomolecular interactions

“…Similar FRET-based approaches have been used to study the kinetics of association of proteins with SNARE complexes [28,29] and the interaction of chromatin remodeling factors with nucleosomes [30]. The obvious advantage of FRET-based methods is that they also allow the study of conformational changes within complexes [4,11,26,[31][32][33].…”

“…The ability to observe biochemical reactions at the level of a single molecule has greatly contributed to our understanding of the molecular mechanisms that define life [1][2][3][4][5][6][7][8][9][10][11]. A major strength of studying processes at the level of individual molecules lies in the direct measurement of distributions of molecular properties, rather than their ensemble averages.…”

“…Single-molecule methods do not only enable the direct observation of binding and unbinding events for a very precise determination of rate constants at a large range of rates and K d 's, but also allow the visualization of various types of heterogeneity in binding properties. Different members of a seemingly homogeneous collection of molecules may exhibit different binding kinetics (so-called static disorder), or individual members of a population may exhibit binding kinetics that change over time (dynamic disorder) [11][12][13][14][15][16]. Characterizing these different phenomena and correlating them with information on structural conformation will increase our understanding of the relationship between activity and structure, an important factor in both binding and enzymatic activity [14,[17][18][19][20][21][22].…”

Fluorescence Molecule Counting for Single-Molecule Studies in Crowded Environment of Living Cells without and with Broken Ergodicity

“…Moving on from time-averaged ensemble measurements to studies on time-averaged single enzyme molecules immobilized on cover slips allows for detection of dynamic disorder, i.e. time-dependent fluctuations in the single-enzyme turnover [ 2 - 4 ].…”

Real-time tracking of single-molecule collagenase on native collagen and partially structured collagen-mimic substrates

“…However, investigating dynamic interactions between individual MMPs and substrates is difficult using conventional fluorescence-based techniques for two main reasons. First, temporal, spatial, and population averaging effects prevent probing of individual biological events 12 with substrates which could be heterogeneous in size and feature a cross-linked structure involving bindings of GPO repeats and fatty acids. 6, 10 Second, signals from the optical techniques are limited by the complete dissociation of fluorophore-quencher units in the substrates regardless of their helicity, which not only preclude the observation of single cleavage events occurring each single-strand, but also interfere with enzymes’ specificity and processivity.…”