Extracting the protein dynamics of bovine serum albumin in the native condition using confocal fluorescent temperature jump

A second-resolved temperature jump (T-jump) system coupled with confocal fluorescent thermometry of 100-μm spatial resolution was employed to unravel the protein dynamics of bovine serum albumin (BSA) at initial temperatures of 25-42 °C. The different evolutions of the tryptophan fluorescence intensity change of the BSA and pure tryptophan upon increasing 5 °C from 25 °C indicated an intrinsic protein dynamics quantified with an apparent rise time of 27 ± 1 s, whereas such a process was not observed at initial temperatures >30 °C. Different heating rates could differentiate the reaction pathways to detour toward reactions possessing different apparent activation energies, successfully explaining the discrepancy of the fluorescence intensity change observed in steady-state and T-jump methods. Moreover, the temperature-dependent steady-state circular dichroism and tryptophan fluorescence spectra were not coincident because they reflected the global and localized structural alterations upon heating, respectively. The combination of the experimental approach and concomitant analysis of the fluorescence evolutions provides a new tool to illustrate the dynamic processes of a big protein upon thermal stimulus.