期刊
ANALYTICAL CHEMISTRY
卷 81, 期 5, 页码 1865-1871出版社
AMER CHEMICAL SOC
DOI: 10.1021/ac802327h
关键词
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资金
- National Eye Institute [T32EY07135, R01-12018, R01-EB353701]
We report the quantitative, label-free analysis of protein-protein interactions in free solution within picoliter volumes using backscatter interferometry (BSI). Changes in the refractive index are measured for solutions introduced on a PDMS microchip allowing determination of forward and reverse rate constants for two-mode binding. Time-dependent BST traces are directly fit using a global analysis approach to characterize the interaction of the small heat-shock protein alpha-Crystallin with two substrates: destabilized mutants of T4 lysozyme and the in vivo target beta B1-Crystallin. The results recapitulate the selectivity of alpha B-Crystallin differentially binding T4L mutants according to their free energies of unfolding. Furthermore, we demonstrate that an alpha A-Crystallin mutant linked to hereditary cataract has activated binding to beta B1-Crystallin. Binding isotherms obtained from steady-state values of the BSI signal yielded meaningful dissociation constants and establishes BSI as a novel tool for the rapid identification of molecular partners using exceedingly small sample quantities under physiological conditions. This work demonstrates that BST can be extended to screen libraries of disease-related mutants to quantify changes in affinity and/or kinetics of binding.
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