File S1 - Ca2+ Binding Enhanced Mechanical Stability of an Archaeal Crystallin

SDS PAGE analysis and Size Exclusion chromatograms; CD spectra; Steady state fluorescence spectra; ITC experimental data; SMFS experimental data obtained using a single cantilever; Peak-wise unfolding forces; Unfolding force histograms from Ca2+ titration experiment; Monte Carlo simulation fits; Table showing the unfolding parameters obtained from Monte Carlo simulations. Table S1. Range of the unfolding rate (ku0) and the distance to the unfolding transition state Δxu by fitting the unfolding force (average), average-SD, and average+SD to Monte Carlo (MC) simulations. Figure S1. Gel electrophoresis results of purified monomer and octamer of M-crystallin. SDS PAGE of M-crystallin monomer (A) and octamer (B) showing bands at ∼11 kDa and ∼90 kDa, respectively. Size exclusion chromatogram of M-crystallin eluted at Superdex 75 and 200 columns for monomer (C) and octamer (D) respectively indicating their high purity level in native conditions (10 mM Tris buffer with 50 mM KCl, pH 7.5). Figure S2. Circular dichroism (CD) spectra of monomer and octamer of M-crystallin. Far-UV CD spectra of M-crystallin monomer (A) and octamer (B). Near-UV CD spectra of M-crystallin monomer (C) and octamer (D). Apo protein spectra are shown in black color and holo protein spectra in grey color. Figure S3. Steady state fluorescence spectra of monomer and octamer of M-crystallin. Emission spectra of M-crystallin apoform monomer (black solid line) and octamer (grey solid line) in native conditions. Emission spectra in denaturing condition (6 M GdnHCl) for monomer (black dashed line) and octamer (grey dashed line). The spectra of holoform were identical. Figure S4. Ca2+ binding measurements using isothermal titration calorimetry (ITC) for M-crystallin monomer (A) and octamer (B). (Top) Reaction heats measured from stepwise calorimetry performed with 5 mM CaCl2 injected against 180 µM M-crystallin in the cell. (Bottom) Binding isotherms are fitted with two-site sequential binding model and results are given in Table 1. Figure S5. Peak-wise unfolding forces of apo and holo protein of M-crystallin at the pulling speed of 1000 nm/sec. There is 30–35 pN enhancement in the mechanical stability of M-crystallin upon Ca2+ binding. The errors are standard deviations. Figure S6. The unfolding force histograms from the pulling experiment done on apo and holo (M-crystallin)8 using the same cantilever, also concur with Fig. 3(B). Figure S7. Histograms of unfolding forces of M-crystallin at different Ca2+ concentrations. The errors indicate standard deviation. Histogram of 30 µM Ca2+ data is plotted with smaller bin-size to show that bimodal distribution is not observed. Figure S8. Monte Carlo simulation fits to speed dependent unfolding forces. Unfolding forces (average), average-SD, and average+SD were separately fitted using Monte Carlo simulations to extract the range of kuo and Δxu. Results are given in Table S1. (DOCX)