Figure 1.

Screening and Validation of Stabilizing Hit Compounds for HMBS

(A) Representative differential scanning fluorimetry (DSF) profiles for HMBS in the absence and presence of hit compounds. The thermal upshift values (ΔT_m) for the compounds are determined from the midpoint denaturation temperatures (T_m at y = 0.5) compared to the control sample with 2% DMSO (T_m = 76.6 ± 0.5°C). The profiles represent recombinant WT-HMBS incubated with the 0.04-mg/mL compound (average concentration 122 μM) in 2% DMSO (red, C5; blue, C6; see the formulas in Table 2) and the control sample without compound, with 2% DMSO (dotted gray). (B) Protection of hit compounds against limited tryptic proteolysis of WT-HMBS. Top: SDS-PAGE showing the effect of the indicated compounds (84 μM and 2% DMSO) on HMBS trypsination. std, low molecular weight standards; control n.t., no trypsin added; control DMSO, HMBS with 2% DMSO and trypsin. Bottom: quantification of the lowest 31.5-kDa band relative to the full-length HMBS at 42.5 kDa. *p < 0.05 and **p < 0.01 for differences compared to the DMSO control, calculated by unpaired two-tailed t test. Data are presented as mean ± SD.