Chain-Folding Initiation Structures in Ribonuclease A: Conformational Analysis of trans-Ac-Asn-Pro-Tyr-NHMe and trans-Ac-Tyr-Pro-Asn-NHMe in Water and in the Solid State

In order to investigate the role of β-bends with non-native trans peptide bonds preceding Pro¹¹⁴ and Pro⁹³ in the mechanism of folding of bovine pancreatic ribonuclease A, we have examined the conformations of the synthetic peptides Ac-Asn-Pro-Tyr-NHMe (I) and Ac-Tyr-Pro-Asn-NHMe (II) in water and in the solid state. These sequences occur at residues 113-115 and 92–94, respectively, in ribonuclease A. Evidence for a significant population of I with a trans-Asn-Pro peptide bond and a β-bend at Pro-Tyr in water includes backbone/backbone and side chain/backbone NOE's, N-H bending frequencies characteristic of a β-bend, and hydrogen bonds detected by solvent spin-saturation transfer measurements involving the TyrNH and NHMe amide protons. Comparison of the Raman spectrum in water with that of the crystal suggests that the major backbone conformation in solution is similar to that in the solid state. Populations of II with a trans-Tyr-Pro peptide bond also have a hydrogen bond involving the NHMe amide. The crystal structure of I (R ≤ 0.064), determined in two different crystalline forms, is a type I β-bend at Pro-Tyr with a trans peptide bond at Asn-Pro and intramolecular hydrogen bonds involving both the TyrNH and NHMe amide protons. The crystal structure of II (R < 0.076) also has a trans peptide bond (at Tyr-Pro), but an extended backbone conformation. The relevance of these results to the mechanism(s) of chain-folding initiation in ribonuclease A is discussed.