Early folding intermediate of ribonuclease A.

Abstract

Pulsed hydrogen exchange (2H-IH) is used to characterize the folding process of ribonuclease A (disulfide bonds intact). The results show one principal early folding intermediate (Ii), which is formed rapidly after the start of folding and whose proton-exchange properties change with the time of folding. AU probes that are hydrogen bonded within the ,8-sheet of native ribonuclease A are protected in II. Thus, the results suggest that the fl-sheet is formed rapidly and cooperatively. The initial protection factors of probes in the fl-sheet are between 10 and 100, but they increase with time of folding and exceed 1000 at 400 msec from the start offolding. Thus, the fl-sheet is only moderately stable when it is first formed, but subsequent events stabilize it, possibly through interactions involving hydrophobic side chains. The large protection factors of the f8-sheet probes in an early folding intermediate are unexpected and remarkable. Probes in the three a-helices are fewer in number and give less accurate data than the f8-strand probes. The folding kinetics expected for a simple sequential model of folding are outlined. An important difference between the observed and predicted behavior is that the early folding intermediate is not fully populated when it is first formed.A recently developed method (1, 2) of studying kinetic folding intermediates uses 2H-1H exchange to pulse label accessible peptide amide protons in folding intermediates. A preliminary study of the folding of RNase A, disulfide bonds intact, has been reported (1). Since then, the complete amide proton assignments of RNase A have been reported (3, 4), and the results, which have been extended, can now be interpreted in structural terms. Twenty-seven amide protons are used as probes of structure in this work: their locations are shown in Fig. 1. All but two are hydrogen bonded and all are located in a-helices or 8-strands; more than half are in the threestranded antiparallel 8-sheet.Our report is focused on early events in the folding process. In kinetic experiments using pulse labeling to measure protection factors, the measurable range is 1-1000. Larger protection factors cannot be quantitated, but ones measured for native proteins are as large as 109 (7). The protection factor is defined as the ratio of two exchange rate constants: k (solvent exposed)/k (observed), where the solvent-exposed rate is measured on a peptide with the same sequence or is calculated from data on model peptides (8). Pulsed hydrogen exchange shows which protons are protected in a folding intermediate and can give their protection factors, but it does not show directly whether protection occurs by hydrogen bonding and it does not reveal hydrogen-bond acceptor groups. The first step in structural interpretation is to compare the pattern of protection results with the structure of native RNase A.Significant changes in methodology have been made since our previous report (1). In that work, introduction of pulse labeling was found to be an important advance over the...

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