Showing papers by "Deepak Sharma published in 2007"

Single-molecule force spectroscopy reveals a mechanically stable protein fold and the rational tuning of its mechanical stability

Abstract: It is recognized that shear topology of two directly connected force-bearing terminal beta-strands is a common feature among the vast majority of mechanically stable proteins known so far. However, these proteins belong to only two distinct protein folds, Ig-like beta sandwich fold and beta-grasp fold, significantly hindering delineating molecular determinants of mechanical stability and rational tuning of mechanical properties. Here we combine single-molecule atomic force microscopy and steered molecular dynamics simulation to reveal that the de novo designed Top7 fold [Kuhlman B, Dantas G, Ireton GC, Varani G, Stoddard BL, Baker D (2003) Science 302:1364-1368] represents a mechanically stable protein fold that is distinct from Ig-like beta sandwich and beta-grasp folds. Although the two force-bearing beta strands of Top7 are not directly connected, Top7 displays significant mechanical stability, demonstrating that the direct connectivity of force-bearing beta strands in shear topology is not mandatory for mechanical stability. This finding broadens our understanding of the design of mechanically stable proteins and expands the protein fold space where mechanically stable proteins can be screened. Moreover, our results revealed a substructure-sliding mechanism for the mechanical unfolding of Top7 and the existence of two possible unfolding pathways with different height of energy barrier. Such insights enabled us to rationally tune the mechanical stability of Top7 by redesigning its mechanical unfolding pathway. Our study demonstrates that computational biology methods (including de novo design) offer great potential for designing proteins of defined topology to achieve significant and tunable mechanical properties in a rational and systematic fashion.

A functional single-molecule binding assay via force spectroscopy

Abstract: Protein–ligand interactions, including protein–protein interactions, are ubiquitously essential in biological processes and also have important applications in biotechnology. A wide range of methodologies have been developed for quantitative analysis of protein–ligand interactions. However, most of them do not report direct functional/structural consequence of ligand binding. Instead they only detect the change of physical properties, such as fluorescence and refractive index, because of the colocalization of protein and ligand, and are susceptible to false positives. Thus, important information about the functional state of protein–ligand complexes cannot be obtained directly. Here we report a functional single-molecule binding assay that uses force spectroscopy to directly probe the functional consequence of ligand binding and report the functional state of protein–ligand complexes. As a proof of principle, we used protein G and the Fc fragment of IgG as a model system in this study. Binding of Fc to protein G does not induce major structural changes in protein G but results in significant enhancement of its mechanical stability. Using mechanical stability of protein G as an intrinsic functional reporter, we directly distinguished and quantified Fc-bound and Fc-free forms of protein G on a single-molecule basis and accurately determined their dissociation constant. This single-molecule functional binding assay is label-free, nearly background-free, and can detect functional heterogeneity, if any, among protein–ligand interactions. This methodology opens up avenues for studying protein–ligand interactions in a functional context, and we anticipate that it will find broad application in diverse protein–ligand systems.