Figure 3. Correlation between T_m of refolded hVDAC-2 and LDAO concentration.

T_m values obtained in the various LDAO concentrations described in this study show a near-exponential increase with LDAO concentration, till ∼65 mM. Increasing in LDAO concentration beyond ∼65 mM does not substantially change the T_m. The T_m of samples that were directly refolded in 5 mM LDAO (labeled ‘Direct’), has been included for comparison, to emphasize the importance of LPR as well as absolute LDAO concentrations during hVDAC-2 refolding. The latter samples exhibit a T_m comparable to those prepared by dilution of the refolding stock to obtain the same LPRs; however, unfolding cooperativity is lost in the ‘Direct’ samples (see inset of Figure 2), emphasizing the importance of absolute LDAO concentration during refolding for protein stability. T_m values were obtained from fits of three independent T-scan unfolding experiments to a two-state equation.