Early folding intermediate of ribonuclease A

Abstract

Pulsed hydrogen exchange (2H-1H) is used to characterize the folding process of ribonuclease A (disulfide bonds intact). The results show one principal early folding intermediate (I1), which is formed rapidly after the start of folding and whose proton-exchange properties change with the time of folding. All probes that are hydrogen bonded within the beta-sheet of native ribonuclease A are protected in I1. Thus, the results suggest that the beta-sheet is formed rapidly and cooperatively. The initial protection factors of probes in the beta-sheet are between 10 and 100, but they increase with time of folding and exceed 1000 at 400 msec from the start of folding. Thus, the beta-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 beta-sheet probes in an early folding intermediate are unexpected and remarkable. Probes in the three alpha-helices are fewer in number and give less accurate data than the beta-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.