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, 81 (5), 1865-71

Free-solution Label-Free Detection of Alpha-Crystallin Chaperone Interactions by Back-Scattering Interferometry

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Free-solution Label-Free Detection of Alpha-Crystallin Chaperone Interactions by Back-Scattering Interferometry

Joey C Latham et al. Anal Chem.

Abstract

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.

Figures

Figure 1
Figure 1
a) Binding of sHSP to their substrate is represented by three coupled equilibria: (1) T4L transition from native (N) to unfolded (U) states, (2) dissociation of the sHSP large oligomer into dimers or tetramers, and (3) formation of the sHSP•T4L complex. HA and LA refer to high affinity and low affinity complexes respectively. Steady-state BSI data (◆) shows that the magnitude of the binding signal increases upon the addition of increasing concentrations of T4L-L99A to a fixed concentration of αB-D3 (7.5 µM). [T4L-L99A] = 1.0, 2.5, 5.0, 7.5, 10, 15, 20, 25, 30, and 40 µM. The linear rise in starting values (■) reflects the response of BSI to increased concentrations of free L99A. c) The slope of the starting values of the traces in c) is identical to that obtained from direct injection of T4L without αB-D3. (d) The calibration curve was used as a baseline subtraction to obtain a corrected steady-state binding trace. Isothermal titration calorimetry (ITC) analysis of αB-D3•T4L-L99A. (e) Heat evolved after each 10 µL injection of T4L-L99A (120 µM) into a reservoir containing 1.4 mL of αB-D3 (12 µM) was detected for 25 injections. f) The area under the curve was extracted and plotted against the molar ratio to obtain a binding isotherm. Non-linear least-squares analysis (red curve) was used to determine thermodynamic parameters.
Figure 2
Figure 2
a) Structure of T4L highlighting the mutation sites. b) Time-dependence of BSI signals following the injection of T4L mutants without sHSP. Increasing T4L concentration shifts the baseline but does not lead to a time-dependent change, ruling out mixing artifacts. c) BSI fringe patterns of the T4L mutants demonstrate BSI is insensitive to differences in their stabilities. A zoomed in region of the interference patterns is shown and compared to a fringe pattern from a buffer solution demonstrating the sensitivity of the instrument to changes in refractive index.
Figure 3
Figure 3
Kinetics of αB-D3 (10 µM) binding to multiple concentrations of T4L-D70N (a) and T4L-L99A-A130S (b) were monitored by BSI (black) with kinetic traces fit via global analysis (red). Analysis of the steady-state data (c) shows that the magnitude of binding as detected by BSI for αB-D3•T4L-L99A-A130S is significantly greater than seen with αB-D3•T4L-D70N. As a control, αB-D3 was assayed against multiple concentrations of WT-T4L, exhibiting no binding across the concentration range. [T4L-D70N] = 1, 2.5, 5, 10, 20, 30, 40, 60, and 90 µM. [T4L-L99A-A130S] = 1, 2.5, 5, 10, 15, 20, 40, 60, 80, and 100 µM.
Figure 4
Figure 4
(a) Interaction of a constant concentration of αA-R49C-crystallin (15 uM) with multiple concentrations of βB1-crystallin at physiologically relevant conditions was detected by BSI. [βB1-crystallin] = 5, 10, 15, 20, 40, 60. Corrected steady-state values (formula image - b) were obtained by background subtraction of a βB1 calibration curve (■ - b). Global analysis of BSI kinetic data was compared to published kinetics from SPR experiments (c).

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