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A functional single-molecule binding assay via force spectroscopy

TitleA functional single-molecule binding assay via force spectroscopy
Publication TypeJournal Article
Year of Publication2007
AuthorsCao, Y, Balamurali, MM, Sharma, D, Li, HB
JournalProceedings of the National Academy of Sciences of the United States of America
Volume104
Pagination15677-15681
Date PublishedOct
ISBN Number0027-8424
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.

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