Nanosecond temperature jump and time-resolved Raman study of thermal unfolding of ribonuclease A.
A 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-microm heat pulse with 9 ns width at 10 Hz was obtained through the two-step stimulated Raman scattering in D(2) 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 897 cm(-1) bands of MoO(4)(2-) ions in an aqueous solution. The T-jump as large as 9 degrees C in 10 ns was attained. The unfolding of bovine pancreatic ribonuclease A was monitored with time-resolved Raman spectra excited at 532 nm. The C-S stretching band of Met residues exhibited 10% change of that expected from the stationary state temperature-difference spectra in the initial 200 ns following T-jump and another 10% in 5 ms. The Raman intensity of SO(4)(2-) ions around 980 cm(-1) increased at 100 micros, presumably due to some conformational changes of the protein around the active site. The S-S stretches and tyrosine doublet displayed little changes within 5 ms. Thus, the conformational changes in the initial step of unfolding are not always concerted.
Yamamoto
K
K
School of Mathematical and Physical Science, The Graduate University for Advanced Studies, Myodaiji, Okazaki 444-8585, Japan.
Mizutani
Y
Y
Kitagawa
T
T
D000818:Animals; D002417:Cattle; D006358:Hot Temperature; D008958:Models, Molecular; D008982:Molybdenum / Q000737:chemistry; D011487:Protein Conformation; D011489:Protein Denaturation / Q000502:physiology; D012259:Ribonuclease, Pancreatic / Q000737:chemistry*; D013059:Spectrum Analysis, Raman; D013431:Sulfates / Q000737:chemistry; D013696:Temperature; D013997:Time Factors
D013431:Sulfates; D008982:Molybdenum; C024687:sodium molybdate(VI); D012259:Ribonuclease, Pancreatic