Synopsis'H-Nmr was used to measure the rate of &-trans interconversion of X-Pro bonds in linear and cyclic oligopeptides. k(cis -trans) = 2.5 X s-l at 25°C was found for the zwitterionic form of H-Ala-Pro-OH, in good agreement with earlier measurements. Replacement of Ala by Phe, Tyr, or Trp resulted in a 10-fold slower interconversion rate, whereas after substitution of Ala by His or Glu, the rate decreased only slightly. Independent of the residues X, the interconversion rate was increased by a factor of ca. 20 when the peptide chain was elongated by addition of Ala to the C-terminal Pro. An additional increase by a factor of 6 was observed when going from the protected linear peptide CF3CO-Gly-Gly-Pro-Ala-OCH3 to the closely related cyclic compound c [-Gly-Gly-Pro-Gly-Ala-1. These data are evaluated with regard to their possible use in future studies on the role of X-Pro &-trans isomerization in the kinetics of protein folding.

SynopsisThe equilibrium between the cis and trans forms of X-Pro peptide bonds can readily be measured in the 13C nmr spectra. In the present paper we investigate how observation of this equilibrium could be used as an nmr probe for conformational studies of flexible polypeptide chains. The experiments include studies by 13C nmr of a series of linear oligopeptides containing different X-L-Pro peptide bonds, with X = Gly, L-Ala, L-Leu, L-Phe, D-Ala, D-Leu, and D-Phe. Overall the study confirms that X-Pro peptide bonds can generally be useful as ':C nmr probes reporting the formation of nonrandom conformations in flexible polypeptide chains. It was found that the cis-trans equilibrium of X-Pro is greatly affected by the side chain of X and the configuration of the a-carbon atom of X. On the basis of these observations some general rules are suggested for practical applications of the X-Pro nmr probes in conformational studies of polypeptide chains.

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SynopsisThe molecular conformations of the linear oligopeptides H-(t,-Ala)Iz -I.-Pro-OH, with n = 1 , 2 , and 3, have been investigated. ':IC nmr observation of the equilibrium between the cis and trans forms of the Ala-Pro peptide bond indicated the occurrence of nonrandom conformations in solutions of these flexible peptides. The formation of the nonrandom species containing the cis form of the Ala-Pro bond was found to depend on the deprotonation of the carboxylic acid group of proline, the solvent, and the ionic strength in aqueous solution. The influence of intramolecular hydrogen bonding on the relative conformational energies of the species containing the cis and trans Ala-Pro peptide bond was studied by comparison of the peptides H-(Ala), -Pro-OH with analogous molecules where hydrogen bond formation was excluded by the covalent structure. In earlier work a hydrogen bond between the protonated terminal carboxylic acid group and the carbonyl oxygen of the penultimate amino acid residue had been suggested to stabilize conformations including trans proline. For the systems described here this hypothesis can be ruled out, since the &:trans ratio is identical for molecules with methyl ester protected and free protonated terminal carboxylic acid groups of proline. Direct evidence for hydrogen bond formation between the deprotonated terminal carhoxylic acid group and the amide proton of the penultimate amino acid residue in the molecular species containing cis proline was obtained from 'H nmr studies. However, the the Ala-Pro bond was not affected by N-methylation of the penultimate amino acid residue, which prevents formation of this hydrogen bond. Overall the experimental observations lead to the conclusion that the relative energies of the peptide conformations including cis or trans proline are mainly determined by intramolecular electrostatic interactions. whereas in the molecules considered, intramolecular hydrogen bonding is a consequence of specific peptide backbone conformations rather than a cause for the occurrence of energetically favored species. Independent support for this conclusion was obtained from model considerations which indicated that electrostatic interactions between the terminal carboxylic acid group and the carbonyl oxygen of the penultimate amino acid residue could indeed account for the observed relative conformational energies of the species containing cis and trans proline. respectively.

A new method for studying membrane transport is presented. High resolution n.m.r. is used to measure the distribution of small molecules between the intracellular and extracellular compartments. The method uses spin-echo techniques and relies on a difference in the magnetic susceptibility of the media inside and outside of cells. It also provides simultaneous information on the metabolic status of the cell. The method is illustrated by a study of alanine and lactate transport in the human erythrocyte.