Screening of folding conditions for hV3 in LDAO micelles. hV3 folding and stability at various protein and LDAO concentrations were screened by changing the DPR. At DPRs <8,500:1, we obtain a large population of folded dimers and higher-order oligomers of hV3. This population of oligomers varies across the preparations. Further, the thermal parameters we measure vary nonlinearly with increasing hV3 concentrations, thus giving rise to experimental artifacts in the analysis. This is demonstrated using the results from screens performed using the C2,8,122,229A variant, as this mutant is moderately stable and has a high aggregation propensity across the series. (A) Effect of protein concentration on structure and thermal stability of hV3 at a constant detergent concentration of 1% LDAO, measured by varying the DPR. Left: The protein exhibits the anticipated β-sheet structure upon folding in LDAO. Here, we observe a considerable decrease in the secondary structure content when the protein concentration is increased (light blue to dark blue). Middle: Dependence of T_m of aggregation on protein concentration at various temperature ramp rates (dark red to light red). Here, we observe a linear decrease in T_m upon increasing the hV3 concentration, at all the ramp rates. The observed linear correlation between protein concentration and T_m suggests that the aggregation can be studied at any of these concentrations without considerable influence from the absolute protein concentration. We have used the lowest protein concentration (red arrow), to minimize the contribution of nonspecific aggregation in our measurements. Right: Correlation between T_m and temperature ramp rate, at a fixed DPR of 8,500:1. We observe a linear increase in T_m at lower ramp rates (<2°C/min); at high ramp rates (>2°C/min), we observe nonlinearity between T_m and temperature ramp rate. The error bars indicate goodness of fit. (B) Effect of protein concentration on structure and thermal stability of hV3 at a constant DPR of ∼8,500:1. Here, both the protein and LDAO concentrations have been proportionately varied from 2.5 to 20 µM and 0.5 to 4%, respectively, so as to maintain a constant DPR. Left: Secondary structure content of hV3 at a constant DPR of ∼8,500:1. The folded protein exhibits similar secondary structure in most of the concentrations (light blue to dark blue), except the lowest LDAO concentration of 0.5%. Middle: Dependence of T_m of aggregation on protein concentration at various temperature ramp rates (dark red to light red). Here, we observe a linear increase in T_m upon increasing both LDAO and hV3 concentration between 0.5 and 2% LDAO. Above 2% LDAO, we observe nonlinearity in T_m and protein concentration at all the ramp rates_. Thus, the LDAO concentration between 0.5 and 2% is sufficient for optimal folding of hV3 and to study protein unfolding and aggregation. We do not observe aggregation >2% LDAO at a temperature ramp rate of 5°C/min; thus, we were not able to derive the T_m of aggregation at higher LDAO concentrations. Right: Correlation between T_m of aggregation and temperature ramp rate at a fixed DPR of ∼8,500:1 (1% LDAO and 5 µM hV3). Here again, we observe a linear correlation between T_m and temperature ramp rate up to 2°C/min; the dependence is nonlinear at faster ramp rates. The error bars indicate goodness of fit. Overall, the data suggest that a moderate protein and detergent concentration can be used to optimally fold hV3 constructs to study protein unfolding and aggregation. We chose a final concentration of 5 µM hV3 in 1% LDAO for our experiments (DPR = 8,500:1), as we obtained a linear dependence of the protein concentration on the T_m and ramp rate (denoted in all the graphs by a red arrow).