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

Elisha Haas, Gaetano T. Montelione, Charles A. McWherter, Harold A. Scheraga

Research output: Contribution to journalArticlepeer-review

23 Scopus citations

Abstract

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.

Original languageEnglish (US)
Pages (from-to)1672-1683
Number of pages12
JournalBiochemistry
Volume26
Issue number6
DOIs
StatePublished - Mar 1 1987
Externally publishedYes

All Science Journal Classification (ASJC) codes

  • Biochemistry

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