Free-solution label-free detection of alpha-crystallin chaperone interactions by back-scattering interferometry

Anal Chem. 2009 Mar 1;81(5):1865-71. doi: 10.1021/ac802327h.


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.

Publication types

  • Research Support, N.I.H., Extramural

MeSH terms

  • Crystallins / chemistry
  • Drug Interactions
  • Interferometry / methods*
  • Molecular Chaperones / chemistry*
  • Mutagenesis, Site-Directed
  • Protein Conformation
  • Protein Folding
  • Staining and Labeling / methods*
  • Structure-Activity Relationship
  • alpha-Crystallins / chemistry*


  • Crystallins
  • Molecular Chaperones
  • alpha-Crystallins