AMIDE PROTON-EXCHANGE USED TO MONITOR THE FORMATION OF A STABLE ALPHA-HELIX BY RESIDUES 3 TO 13 DURING FOLDING OF RIBONUCLEASE-S

The known exchange rates of individual amide protons in the S-peptide moiety of tRNase S were used to determine when during folding the .alpha.-helix formed by residues 3-13 becomes stable. The method is based on pulse-labeling with [3H]H2O during the folding followed by an exchange-out step after folding that removes 3H from all amide protons of the S-peptide except from residues 7-14, after which S-peptide is separated rapidly from S-protein by high performance liquid chromatography. The slow-folding species of unfolded RNase S are studied. Folding takes place in strongly native conditions (pH 6.0 10.degree. C). The 7 H-bonded amide protons of the 3-13 helix become stable to exchange at a late stage in folding at the same time as the tertiary structure of RNase S is formed, as monitored by Tyr absorbance. At this stage in, folding, the isomerization reaction that creates the major slow-folding species was not yet reversed. The result for the 3-13 heliz is consistent with the finding of Labhardt who has studied the kinetics of folding of RNase S at 32.degree. C by fast circular dichroism. He finds the dichroic change expected for formation of the 3-13 helix occurring when the tertiary structure is formed. Protected amide protons are found in the S-protein earlier in folding. Formation or stabilization of this folding intermediate depends upon S-peptide; the intermediate is not observed when S-protein folds alone, and folding of S-protein is twice as slow in the absence of S-peptide. Although S-peptide combines with S-protein early in folding and is needed to stabilize an S-protein folding intermediate, the S-peptide helix does not itself become stable until the tertiary structure of RNase S is formed.