DETECTION OF AN EARLY INTERMEDIATE IN THE FOLDING OF RIBONUCLEASE-A BY PROTECTION OF AMIDE PROTONS AGAINST EXCHANGE

A new method is presented for detecting structural intermediates at early stages of protein folding. The principle is to label with 3H the exchangeable amide protons of the unfolded protein and then to choose refolding conditions (pH, temperature) where rapid exchange-out will occur unless folding intermediates protect some protons. The label which would be retained after folding if there were no folding intermediates is calculated and subtracted to give the number of protected protons. Folding is allowed to go to completion and the number of protected protons which are stable to exchange for 6 to 13 hours after folding is determined. The slow-folding class of unfolded ribonuclease A has been chosen for study. The folding conditions are: 10 °C, pH 6 to 8, 0.2 to 2.5 m-guanidine · HCl. A large fraction ( 1 3) of the stable protons of native RNAase A is protected during folding. This fraction drops rapidly with increasing guanidine · HCl molarity as folding intermediates are destabilized. At 2.5 m-guanidine · HCl, pH 6, only 1.5 protons are protected although folding goes to completion and there is little effect of guanidine · HCl on the exchange kinetics of the stable protons in native RNAase A. This result provides a negative control. A second control is given by RNAase S, for which no protected protons are found after folding. We conclude that the S-peptide moiety of RNAase A is needed for stability of the early folding intermediate. The number of protected protons also decreases with pH in the range pH 6 to 8: this result is expected since the exchange step is base-catalyzed while the folding kinetics are independent of pH between 6 and 8. There are two conclusions about the kinetic mechanism of folding. 1. (1) The structural basis for protection against exchange appears to be formation of an H-bonded structure, since exclusion of water from the tertiary structure occurs later, as monitored by tyrosine absorbance. It appears that at least part of the H-bonded backbone of RNAase A is formed at an early stage in folding. The intermediate is stabilized by the S-peptide moiety of RNAase A. 2. (2) Folding of RNAase A is a sequential process, with kinetically observable intermediates, in these conditions. © 1979.