Kinetics of hydrogen bond breakage in the process of unfolding of ribonuclease A measured by pulsed hydrogen exchange.
A sensitive test for kinetic unfolding intermediates in ribonuclease A (EC 3.1.27.5) is performed under conditions where the enzyme unfolds slowly (10 degrees C, pH 8.0, 4.5 M guanidinium chloride). Exchange of peptide NH protons (2H-1H) is used to monitor structural opening of individual hydrogen bonds during unfolding, and kinetic models are developed for hydrogen exchange during the process of protein unfolding. The analysis indicates that the kinetic process of unfolding can be monitored by EX1 exchange (limited by the rate of opening) for ribonuclease A in these conditions. Of the 49 protons whose unfolding/exchange kinetics was measured, 47 have known hydrogen bond acceptor groups. To test whether exchange during unfolding follows the EX2 (base-catalyzed) or the EX1 (uncatalyzed) mechanism, unfolding/exchange was measured both at pH 8.0 and at pH 9.0. A few faster-exchanging protons were found that undergo exchange by both EX1 and EX2 processes, but the 43 slower-exchanging protons at pH 8 undergo exchange only by the EX1 mechanism, and they have closely similar rates. Thus, it is likely that all 49 protons undergo EX1 exchange at the same rate. The results indicate that a single rate-limiting step in unfolding breaks the entire network of peptide hydrogen bonds and causes the overall unfolding of ribonuclease A. The additional exchange observed for some protons that follows the EX2 mechanism probably results from equilibrium unfolding intermediates and will be discussed elsewhere.
Kiefhaber
T
T
Department of Biochemistry, Stanford Medical Center, CA 94305-5307, USA.
Baldwin
R L
RL
D002942:Circular Dichroism; D019791:Guanidine; D006146:Guanidines; D006860:Hydrogen Bonding; D007700:Kinetics; D011487:Protein Conformation; D011489:Protein Denaturation; D012259:Ribonuclease, Pancreatic / Q000737:chemistry* / Q000378:metabolism; D013997:Time Factors
D006146:Guanidines; D012259:Ribonuclease, Pancreatic; D019791:Guanidine