A structural and biochemical approach to effect of temperature and pH on the Molten Globule phenomenon in a thermophilic protease: Molecular dynamics simulation, fluorescence spectroscopy and circular dichroism.

• Thermostable serine protease showed a maximum intrinsic fluorescence in 335 nm and a blue shift in 510 nm for extrinsic fluorescence. • It was suggested that the protein transitions probably to an MG state at pH 3. • Thermostable serine protease showed a little shift in the MG state intermediate at pH 3 in far-UV. • Maximum decreases was observed in 228 to 300 nm for UV-near CD. Proteins in the molten globule (MG) stage have a reasonably stable secondary structure content, but changes in side-chain packing cause a noticeable decrease in the number of tertiary structures. Here, we investigated serine protease as a suitable model to study the folding route and characteristics of a protein in the MG state. Upon induction with IPTG, the protein exhibited a molecular weight of 42 kDa and achieved a final concentration of 1.7 mg/mL after the concentration process. Serine protease's intrinsic and extrinsic fluorescence was examined in the pH range of 1–12 using fluorescence spectroscopy with maximum intrinsic fluorescence in 335 nm and a blue shift in 510 nm for extrinsic fluorescence. It was suggested that the protein transitions probably to an MG state at pH 3. Evidences showed that serine protease's secondary structure using far-UV circular dichroism (CD) showed a little shift in the MG state intermediate, pH 3, relative to the original state, pH 8, which is typical for MG. Maximum decreases was observed in 228 to 300 nm for UV-near CD. Also revealed a notable alteration in the pH 3 of the enzyme compared to pH 8, which is the pH at which the protein takes on its natural structure. By using acrylamide to quench the fluorescence, the Stern-Volmer constant (KSV) was determined at pH values of 8 and 3, yielding values of 20 and 14 M −1, respectively. This is likely correlated with an increase in the enzyme's molecular volume in the MG state. Molecular dynamics (MD) simulation showed that although the thermal stability of this enzyme at 90° was confirmed in the Root-Mean-Square-Deviation (RMSD), Root-Mean-Square-Fluctuation (RMSF), Radius of gyration (Rg) as well as Solvent-Accessible-Surface-Area (SASA) graphs, but pH 3 may significantly disturb the tertiary structure and secondary structure. These in-silico results may also be analyzed in line with the occurrence of MG at pH 3. [ABSTRACT FROM AUTHOR]