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A conformational analysis of gramicidin S-A by nuclear magnetic resonance.

1968; National Academy of Sciences; Volume: 61; Issue: 2 Linguagem: Inglês

10.1073/pnas.61.2.734

1091-6490

A. Stern, William A. Gibbons, Lyman C. Craig,

Microbial Natural Products and Biosynthesis

Currently there is great interest in the precise conformation of complicated natural products in solution. Nuclear magnetic resonance (NiVIR) has great promise in this connection and has made possible a high degree of sophistication in the conformational analysis of the steroids. However, less progress has been made with peptides. In this paper, we wish to report our analysis of 100and 220-AMc N1\MIR spectra of the cyclic antibiotic decapeptide gramicidin S-A in solution. Methods.-Proton magnetic resonance spectra were obtained on Varian Associates high-resolution spectrometers operating at 100 and 220 Mc. The internal reference was tetramethylsilane (TMS), which appears at 0 ppm in all spectra. Sample colncentrations were 3-6%o (gm/100 ml). The normal operating temperature was 320. Gramicidin S-A was isolated from a crude preparation by countercurrent distributionib and lyophilized in glacial acetic acid. Deuterated dimethylsulfoxide (DMS0) and deuterated methanol (CD30D) were purchased from NMR specialties. DMSO-d6 was dried when necessary over calcium hydride. Results and Discussion. A twofold axis of molecular symmetry in the crystal has been demonstrated for gramicidin-S by X-ray crystallography.2 From the known amino acid sequence,3' 4 it seemed likely that this conformation would hold in solution; this is confirmed by the NMIR results reported here. Ten amino acid residues, two of each kind, are present. The strong single resonance band at 7.3 ppm in CD30D (and in DMSO if the partially interfering amide signal is subtracted, see later) must result from the ten aromatic protons of the two phernylalanines. With this as a basic inference, integration under the peak areas assigned to amide bands then corresponds to two protons for each of four regions, although these areas are not all clearly separated by a single solvent and temperature condition. Their assignment to a particular amino acid residue is made possible by decoupling and deuterium exchange experiments as outlined below. The assignments of the proton chemical shifts in the gramicidin S-A spectra are based on the following: (a) comparison with NMVIR spectra of constituent amino acids and known peptides; (b) spectra taken on instruments of varying field strengths (60, 100, and 220 Mc); (c) spectra taken in solvents that promote and solvents that prohibit proton exchange; (d) double resonance experiments; and (e) variable temperature spectra. Decoupling experiments were necessary in order (a) to show that the splitting of the amide proton peaks was due to spin-spin interaction and not to chemical shifts (a fact also proved by the invariability of the amide coupling constants in both the 100and 220-Mic spectra) and (b) to determine which Ca, C:, and NH protons are coupled together. The coupling constants between these types of protons will be designated Ja,3 and JNC. A knowledge of these should define the stereochemistry around the C -C and N-C bonds.