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Observation of the closing of individual hydrogen bonds during TFE–induced helix formation in a peptide

¹ Department of Structural Biology, Biozentrum, University of Basel, Basel CH-4056, Switzerland
² Department of Molecular and Cell Biology, University of Connecticut, Storrs, Connecticut 06269-3125, USA

Reprint requests to: Stephan Grzesiek, Department of Structural Biology, Biozentrum, University of Basel, Klingelbergstr. 70, Basel CH-4056, Switzerland; e-mail: Stephan.Grzesiek{at}unibas.ch; fax: 41-61-2672109 or Andrei Alexandrescu, Department of Molecular and Cell Biology, University of Connecticut, 75 North Eagleville Road, U-3125, Storrs, CT 06269-3125, USA; e-mail: andrei{at}uconnvm.uconn.edu.

Helix formation of an S-peptide analog, comprising the first 20 residues of Ribonuclease A and two additional N-terminal residues, was studied by measuring hydrogen bond (H-bond) ^h3J_NC' scalar couplings as a function of 2,2,2-trifluoroethanol (TFE) concentration. The ^h3J_NC' couplings give direct evidence for the closing of individual backbone N-H•••O = C H-bonds during the TFE-induced formation of secondary structure. Whereas no ^h3J_NC' correlations could be detected without TFE, -helical (i,i +4) H-bond correlations were observed for the amides of residues A5 to M15 in the presence of TFE. The analysis of individual coupling constants indicates that -helix formation starts at the center of the S-peptide around residue E11 and proceeds gradually from there to both peptide ends as the TFE concentration is increased. At 60% to 90% TFE, well-formed -helical H-bonds were observed for the amides hydrogens of residues K9 to Q13, whereas H-bonds of residues T5 to A8, H14, and M15 are affected by fraying. No intramolecular backbone H-bonds are present at and beyond the putative helix stop signal D16. As the ^h3J_NC' constants represent ensemble averages and the dependence of ^h3J_NC' on H-bond lengths is very steep, the size of the individual ^h3J_NC' coupling constants can be used as a measure for the population of a closed H-bond. These individual populations are in agreement with results derived from the Lifson-Roig theory for coil-to-helix transitions. The present work shows that the closing of individual H-bonds during TFE-induced helix formation can be monitored by changes in the size of H-bond scalar couplings.

Keywords: S-peptide; RnaseA; J-coupling; NMR; scalar coupling; protein folding

Abbreviations: TFE, 2,2,2-trifluoroethanol • RNaseA, Ribonuclease A • S-peptide, N-terminal fragment of Ribonuclease A • H-bond, hydrogen bond.

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