Free-solution label-free detection of alpha-crystallin chaperone interactions by back-scattering interferometry.
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 BSI 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 betaB1-Crystallin. The results recapitulate the selectivity of alphaB-Crystallin differentially binding T4L mutants according to their free energies of unfolding. Furthermore, we demonstrate that an alphaA-Crystallin mutant linked to hereditary cataract has activated binding to betaB1-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 BSI can be extended to screen libraries of disease-related mutants to quantify changes in affinity and/or kinetics of binding.
Latham
Joey C
JC
Department of Chemistry and The Vanderbilt Institute for Chemical Biology, Vanderbilt University, VU Station B 351822, Nashville, Tennessee 37235-1822, USA.
Stein
Richard A
RA
Bornhop
Darryl J
DJ
Mchaourab
Hassane S
HS
D003459:Crystallins / Q000737:chemistry; D004347:Drug Interactions; D007368:Interferometry / Q000379:methods*; D018832:Molecular Chaperones / Q000737:chemistry*; D016297:Mutagenesis, Site-Directed; D011487:Protein Conformation; D017510:Protein Folding; D013194:Staining and Labeling / Q000379:methods*; D013329:Structure-Activity Relationship; D038201:alpha-Crystallins / Q000737:chemistry*
D003459:Crystallins; D018832:Molecular Chaperones; D038201:alpha-Crystallins