Hydrogen exchange in thermally denatured ribonuclease A

Journal Article · Tue Oct 15 00:00:00 EDT 1991 · Biochemistry; (United States)

DOI:https://doi.org/10.1021/bi00105a014· OSTI ID:5821012

Robertson, A D; Baldwin, R L ^[1]

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1. Stanford Univ., CA (United States)

Hydrogen exchange has been used to test for the presence of nonrandom structure in thermally denatured ribonuclease A (RNase A). Quenched-flow methods and 2d {sup 1}H NMR spectroscopy were used to measure exchange rates for 36 backbone amide protons (NHs) at 65C and at pH* (uncorrected pH measured in D{sub 2}O) values ranging from 1.5 to 3.8. The results show that exchange is approximately that predicted for a disordered polypeptide, the authors thus are unable to detect any stable hydrogen-bonded structure in thermally denatured RNase A. Two observations suggest, however, that the predicted rates should be viewed with some caution. First, they discovered that one of the approximations made by Molday et al, that exchange for valine NHs is similar to that for alanine NHs, had to modified; the exchange rates for valine NHs are about 4-fold slower. Second, the pH minima for exchange tend to fall at lower pH values than predicted, by as much as 0.45 pH units. The origin of the disagreement between predicted and observed pH minima is unknown but may be the high net positive charge on these proteins at low pH. This spectrum is reduced to a low level by adding guanidine hydrochloride. The nature of the residual structure responsible for this spectrum is not known. The results show that it is not stable helix formation by the three {alpha}-helices of native ribonuclease A, which would give measurable protection against amide proton exchange.

• OSTI ID: 5821012
• Journal Information: Biochemistry; (United States), Vol. 30:41; ISSN 0006-2960
• Country of Publication: United States
• Language: English

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