Local Structure in a Tryptic Fragment of Performic Acid Oxidized Ribonuclease a Corresponding to a Proposed Polypeptide Chain-Folding Initiation Site Detected by Tyrosine Fluorescence Lifetime and Proton Magnetic Resonance Measurements

The effects of proline and X–Pro peptide bond conformations on the fluorescence properties of tyrosine in peptides corresponding to parts of a proposed chain-folding initiation site in bovine pancreatic ribonuclease A are examined by time-resolved and steady-state fluorescence spectroscopy. In peptides with Tyr-Pro sequences, the conformational constraints of proline on a preceding residue result in significant fluorescence quenching for both trans and cis peptide bond conformations. Small peptides containing Pro-Tyr sequences, on the other hand, do not exhibit fluorescence quenching compared to Ac-Tyr-NHMe. Studies of fluorescence decay in the tryptic fragment of performic acid oxidized ribonuclease corresponding to residues 105–124 (i.e., O-T-16) demonstrate the presence of at least two environments of the single tyrosine chromophore (in the sequence Asn¹¹³-Pro¹¹⁴-Tyr¹¹⁵). In these two (ensemble-averaged) environments, tyrosine has shorter and longer lifetimes, respectively, than in Ac-Tyr-NHMe. The fluorescence heterogeneity in O-T-16 does not correlate with X–Pro cis/trans conformational heterogeneity that can be detected by nuclear magnetic resonance (NMR) spectroscopy. Instead, the fluorescence heterogeneity in O-T-16 arises from the presence of multiple conformations with the same X-Pro peptide bond conformations which interconvert rapidly on the ¹H NMR time scale (τ ≪ 1 ms) but are distinguishable on the fluorescence lifetime time scale (τ ≥ 1 ns). From comparisons with the tyrosine fluorescence decay of smaller synthetic peptides, it is concluded that the long-lifetime tyrosine fluorescence component of O-T-16 arises from interactions involving residues outside the Asn¹¹³-Pro¹¹⁴-Tyr¹¹⁵-Val¹¹⁶-Pro¹¹⁷ sequence, which either stabilize particular local conformations in the vicinity of Tyr¹¹⁵ or act directly to protect Tyr¹¹⁵ from efficient fluorescence quenching. The short-lifetime component of O-T-16 is also observed for the pentapeptide Ac-Asn-Pro-Tyr-Val-Pro-NHMe. The data provide evidence for a nonrandom polypeptide conformation of O-T-16 under conditions of solvent pH and temperature at which the complete disulfide-intact ribonuclease molecule is fully folded. Implications of this work for the interpretation of fluorescence-detected unfolding experiments are discussed.