Temperature-jump NMR study of protein folding: ribonuclease A at low pH.
The kinetic process of folding of bovine pancreatic ribonuclease A in a 2H2O environment at pH 1.2 was examined by a recently developed temperature-jump NMR method (Akasaka et al., (1990) Rev. Sci. Instrum. 61, 66-68). Upon temperature-jump down from 45 degrees C to 29 degrees C, which was attained within 6 s, the proton NMR spectral changes were followed consecutively in time intervals of seconds. There was a rapid spectral change, which was finished within the jump period, followed by a much slower process which lasted for a minute or longer. Rates of the slower process were measured at different positions of the polypeptide chain as intensity changes of individual His and Tyr proton signals of the folded conformer and as intensity changes of aliphatic and His protons of the unfolded conformer. Most of these rates coincided with each other within experimental error with an average value of 2.8 x 10(-2) s-1. The result gave clear experimental evidence that the slow folding of RNase A at low pH is a cooperative process involving most regions of the molecule, not only thermodynamically, but kinetically as well.
Akasaka
K
K
Department of Chemistry, Faculty of Science, Kyoto University, Japan.
Naito
A
A
Nakatani
H
H
D000818:Animals; D002417:Cattle; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D009682:Magnetic Resonance Spectroscopy / Q000379:methods*; D011487:Protein Conformation; D011489:Protein Denaturation; D012259:Ribonuclease, Pancreatic / Q000737:chemistry*; D013696:Temperature; D013816:Thermodynamics
D012259:Ribonuclease, Pancreatic