PMC

(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 ( - 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).

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.

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.

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.