Characterization of alpha-helix structures in polypeptides, revealed by C-13=O center dot center dot center dot H-N-15 hydrogen bond lengths determined by C-13 REDOR NMR

We have determined accurate C-13-N-15 interatomic distances of mono-dispers ed sequential oligopeptides, [1-C-13]Leu(8) [N-15]Ala(12)-labeled (Phe-Leu- Ala)(6), and a variety of its derivatives in which Leu, at the residue 11, was replaced by Ala, Phe, Gly or D-Ala, by means of rotational echo double resonance (REDOR) experiments. In order to minimize a plausible contributio n from the peak intensities of natural abundant signals from amino-acid res idues other than labeled, C-13 NMR signals from such contributions were sub tracted from the full echo amplitude of the doubly labeled spectra as refer ence. We have determined the interatomic distance as 4.5 +/- 0.1 A for five kinds of sample examined without any significant deviation among them. Bec ause [1-C-13]Leu(8) and [N-15]Ala(12) form C=(OHN)-H-... hydrogen bond in t he case of or-helical structure, this finding evidently showed the presence of a-helix and is consistent with the data from the conformation-dependent C-13 NMR chemical shifts of corresponding C-13 NMR signals. It turned out, therefore, that characterization of the a-helix structure based on REDOR e xperiment is straightforward and practical, if careful setting of experimen tal conditions is achieved to arrive at the meaningful data. It is also pro ved that no correction from the REDOR effect from the neighboring chains is necessary as far as alpha -helix structures are concerned, because fully a nd 30% labeled samples gave rise to identical REDOR results. Further, it is concluded that unusual displacement of 15N chemical shifts present in the sequential oligopeptide should be ascribed to a contribution other than any modified alpha -helical structures. (C) 2001 Elsevier Science B.V. All rig hts reserved.