A functional single-molecule binding assay via force spectroscopy

@article{citeulike:1942977, abstract = {Proteinligand interactions, including proteinprotein 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 proteinligand 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 proteinligand 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 proteinligand 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 proteinligand interactions. This methodology opens up avenues for studying proteinligand interactions in a functional context, and we anticipate that it will find broad application in diverse proteinligand systems. 10.1073/pnas.0705367104}, author = {Cao, Yi and Balamurali, M. M. and Sharma, Deepak and Li, Hongbin }, citeulike-article-id = {1942977}, doi = {10.1073/pnas.0705367104}, journal = {Proceedings of the National Academy of Sciences}, keywords = {afm, binding, clip2, design, experiment, ligand, protein}, month = {October}, number = {40}, pages = {15677--15681}, posted-at = {2007-11-20 11:19:21}, priority = {2}, title = {A functional single-molecule binding assay via force spectroscopy}, url = {http://dx.doi.org/10.1073/pnas.0705367104}, volume = {104}, year = {2007} }

TY - JOUR ID - citeulike:1942977 L3 - citeulike-article-id:1942977 TI - A functional single-molecule binding assay via force spectroscopy JF - Proceedings of the National Academy of Sciences VL - 104 IS - 40 SP - 15677 EP - 15681 N2 - Proteinligand interactions, including proteinprotein 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 proteinligand 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 proteinligand 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 proteinligand 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 proteinligand interactions. This methodology opens up avenues for studying proteinligand interactions in a functional context, and we anticipate that it will find broad application in diverse proteinligand systems. 10.1073/pnas.0705367104 KW - afm KW - binding KW - clip2 KW - design KW - experiment KW - ligand KW - protein AU - Cao, Yi AU - Balamurali, MM AU - Sharma, Deepak AU - Li, Hongbin PY - 2007/10/02/ UR - http://dx.doi.org/10.1073/pnas.0705367104 ER -