Nanosecond Temperature Jump and Time-Resolved Raman Study of Thermal Unfolding of Ribonuclease A

AbstractA nanosecond temperature jump (T-jump) apparatus was constructed and combined with time-resolved Raman measurements to investigate thermal unfolding of a protein for the first time. The 1.56-μm heat pulse with 9ns width at 10Hz was obtained through the two-step stimulated Raman scattering in D2 gas involving seeding and amplification. To achieve uniform temperature rise, the counter-propagation geometry was adopted for the heat pulse. The temperature rise was determined by anti-Stokes to Stokes intensity ratios of the 317 and 897cm−1 bands of MoO42− ions in an aqueous solution. The T-jump as large as 9°C in 10ns was attained. The unfolding of bovine pancreatic ribonuclease A was monitored with time-resolved Raman spectra excited at 532nm. The C-S stretching band of Met residues exhibited 10% change of that expected from the stationary state temperature-difference spectra in the initial 200ns following T-jump and another 10% in 5ms. The Raman intensity of SO42− ions around 980cm−1 increased at 100μs, presumably due to some conformational changes of the protein around the active site. The S–S stretches and tyrosine doublet displayed little changes within 5ms. Thus, the conformational changes in the initial step of unfolding are not always concerted