A salt bridge stabilizes the helix formed by isolated C-peptide of RNase A.

Abstract

C-peptide, which contains the 13 NH2-terminal residues of RNase A, shows partial helix formation in water at low temperature (1C, pH 5, 0.1 M NaCI), as judged by CD spectra; the helix is formed intramolecularly [Brown, J. E. & Klee, W. A. (1971) Biochemistry 10, 470-476]. We find that helix stability depends strongly on pH: both a protonated histidine (residue 12) and a deprotonated glutamate (residue 9 or 2 or both) are required for optimal stability. This information, together with model building, suggests that the salt bridge Glu-9 His-12+ stabilizes the helix. Formationofthehelixisenthalpydriven [van'tHoff AH, -16kcal/ mol (1 cal = 4.18 J)] and the helix is not observed above 30C.Proton NMR data indicate that several side chains adopt specific conformations as the helix is formed. These results have two implications for the mechanism of protein folding. First, they indicate that short at-helices, stabilized by specific side-chain interactions within the helix, can be stable enough in water to function as folding intermediates. Second, they suggest that similar experiments with peptides of controlled amino acid sequence could be used to catalogue the intrahelix interactions that stabilize or destabilize a-helices in aqueous solution. These data might provide the code relating amino acid sequence to the locations of a-helices in proteins.Short a-helices, of the size range usually found in globular proteins (6-20 residues), are highly unstable in water in the absence of specific stabilizing interactions, according to data obtained with random copolymers by the "host-guest" technique (1). For short helices (n < 20) and for o << 1, the ratio ofhelical to nonhelical residues depends on the quantity 0,rn-l/(S -1)2 (see Note Added in Proof), where o is the helix nucleation constant, s is an average stability constant for one residue, and n is the number of H-bonded residues in the helix. Values of s vary from 0.5 to 1.3 for different amino acid residues at 0-60TC, while o is estimated to be~-'10`(1), so that the fraction of molecules in helical form is expected to be 10`for a short helix. Most studies of helix formation by protein fragments agree with this deduction. The three cyanogen bromide peptides of sperm whale myoglobin give CD spectra that indicate that very little a-helix is present in water at 250C, although partly helical spectra are obtained in 95% methanol (2). The conclusion has been drawn that tertiary interactions are needed to stabilize the a-helices of globular proteins in water.An exception was found by Brown and Klee (3): C-peptide of RNase A has a CD spectrum indicative of partial helix formation in water at 10C. They found that the helix forms monomolecularly, is unstable at 260C, and is unstable in deionized water. Molecular weight measurements by sedimentation equilibrium showed that C-peptide is monomeric at concentrations up to 1 mM under conditions in which helix formation occurs (10C; ionic strength, 0.1 M). Freedom from aggregation at moderate concentration is an important prop...

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β‐strand interactions at the domain interface critical for the stability of human lens γD‐crystallin

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