Single-Molecule Study of Protein−Protein Interaction Dynamics in a Cell Signaling System

Abstract

We report a study on protein−protein noncovalent interactions in an intracellular signaling protein complex, using single-molecule spectroscopy and molecular dynamics (MD) simulations. A Wiskott−Aldrich Syndrome Protein (WASP) fragment that binds only the activated intracellular signaling protein Cdc42 was labeled with a novel solvatochromic dye and used to probe hydrophobic interactions significant to Cdc42/WASP recognition. The study shows static and dynamic inhomogeneous conformational fluctuations of the protein complex that involve bound and loosely bound states. A two-coupled, two-state Markovian kinetic model is proposed for the conformational dynamics. The MD simulations explore the origin of these conformational states and associated conformational fluctuations in this protein−protein interaction system.

Multiple-state reactions between the epidermal growth factor receptor and Grb2 as observed by using single-molecule analysis

“…To realize this mechanism, proteins would require a conformation memory after dissociation. When time constants for conformational transition of the binding site are comparable to that of association reaction, reaction memory can be induced due to dynamic disorder (20)(21)(22)(23). In fact, the existence of reaction memory was suggested by non-Markovian function analysis of the single-molecule reaction trajectories (SI Fig.…”

“…Single-molecule measurement is a powerful experimental technique used to study the details of complex kinetics. Measurements of the distributions and temporal fluctuations in the duration of individual molecular recognition processes using the single-molecule technique have revealed the static and dynamic disorders (20)(21)(22)(23) and memory landscapes (24,25) of enzymatic reactions, as well as dynamic polymorphisms of protein structures (26)(27)(28). In living cells, single-molecule measurements have even been used to clarify spatially inhomogeneous kinetics in receptor-ligand (29) and protein-protein (30) dissociations, and to identify previously unknown reaction intermediates (31).…”

Single‐Molecule Detection and Manipulation in Nanotechnology and Biology

“…For instance, heterogeneous rotational dynamics and lifetimes of single molecules in polymer films can be used to investigate molecular environments present within different regions of the film [48,49]. Sensitive to changes in hydrophobicity, a new solvatochromic dye covalently bound to a polypeptide, for example, was used to monitor protein-protein interactions at the single-molecule level [50]. Inherent versatility, fast time resolution, and high SNR will continue to bolster the use of confocal optics in SMS; however, in contrast to epifluorescence and TIRF designs, which can study multiple point sources at once, confocal detection can only detect point source objects one at a time.…”

Ensemble QSAR: A QSAR method based on conformational ensembles and metric descriptors

“…It has been widely recognized that knowledge about the static structure alone is not enough to probe real-time peptide functions and that conformational changes and ordered-disordered structure transitions, 31,32 play vital roles in determining peptide functions. Thus, peptides serve as ideal candidates to test and validate the eQSAR methodology.…”