Carbon-13 NMR spectroscopy of [20%-1,2-13C2-Gly6]-bradykinin. Role of serine in reducing structural heterogeneity

Abstract

Substitution of a glycine residue for the serine residue at position six in the nonapeptide bradykinin leads to a dramatic increase in the cis/trans ratio about the sixth peptide bond (~38% cis). Decreases in the cis/trans ratio determined by NMR in 1 M NaC104 and in 60% ,2CH30H have been correlated with decreases in the 219-nm CD band. Chemical shifts of the 20% 13C-labeled Gly6 residue exhibit a very different pattern from that observed in the dipeptide Gly-L-Pro; the cis-trans shift difference is ~0 ppm for the glycine carbonyl resonances and ~1.1 ppm for the glycine methylene resonances. In contrast, the cisand rrani-glycine carbonyl resonances are resolved in 60% methanol and in 1 M NaCKAi. A series of model peptides has been examined to determine the structural factors leading to the observed shift pattern. These studies indicate the importance of Phe8 both in determining the cis/trans ratio and in producing the observed chemical shifts. Glycine resonances in the tripeptide Gly-Pro-Phe are qualitatively similar to those observed in [Gly6]-bradykinin. The most probable explanation of these data is a favorable hydrophobic/solvent-excluding Pro-Phe association in the cis peptide, which leads to a rotation about the Pro C"carbonyl bond, placing the carbonyl oxygen in close proximity to the Gly6 methylene carbon. In the trans peptide, electrostatic repulsion between the Gly and Pro carbonyl oxygens opposes this effect. Significant differences in the cis/trans ratios between bradykinin and [Gly6]-bradykinin have prompted careful bioassay studies, which show the potency of [Gly6]-bradykinin to be only 50-70% that of bradykinin, although at saturating levels of peptide the responses are within experimental error. These data provide clues about the functional role of serine in bradykinin.

Non-Statistical Label Distribution in Biosynthetic 13C Enriched Amino Acids

“…However, the starting material is never isotopically pure, so that the resulting compounds such as amino acids, sugars, fatty acids, and even proteins and viruses must also be only partially enriched. It has been assumed in the past that isotopically labelled compounds of biosynthetic origin i) are statistically enriched, and ii) show a uniform enrichment factor [1][2][3].…”

Calcium‐binding properties and molecular organization of bradykinin

“…The presence of three prolines in the sequence is expected to yield structural heterogeneity in solution, because the cis trans interconversion at each X–Pro bond may provide up to eight stable isomers, depending on environmental parameters. Several CD and NMR investigations in a number of solvent systems have occasionally detected transient ordered structures, similar to γ turns and β turns [6–23]; however, the net result of a random coil peptide was obtained in all cases due to the fast rate of exchange between the extended structures. Such a high degree of flexibility suggests that binding to the receptor implies the selection of a particular structure from among the number of slowly or rapidly interconverting ones in solution.…”

Bradykinin and its Gly6 analog are substrates of cyclophilin: a fluorine-19 magnetization transfer study

“…The substitution of a glycine residue for Ser6 of bradykinin significantly increases the cis/trans ratio about the sixth peptide bond from ~0.1 to ~0.4 (London et al, 1979). The physical basis for this effect has been analyzed in detail on the basis of chemical shift, coupling constant, and relaxation data of model peptides and results primarily from an unfavorable interaction between the Phe8 side chain and the Gly6 carbonyl oxygen in the trans configuration (Anteunis et al, 1981).…”

“…Arg-Pro-Pro-Gly-Phe-Ser-Pro-Phe-Arg) and the test peptides which have been used to study cyclophilin-catalyzed cis/trans isomerization (Fischer et al, 1984(Fischer et al, , 1989aTakahashi et al, 1989;Harrison & Stein, 1990), it was of interest to determine whether this biologically active peptide is also a substrate for cyclophilin. We have previously noted that, despite a strong preference for the trans conformation of all X-Pro bonds in bradykinin, a large increase in the cis/trans ratio of the sixth peptide bond, in addition to a small decrease in activity, results from the substitution of a Gly6 residue for the native Ser6 (London et al, 1979). On this basis, we have suggested that the predominant trans conformer is most probably the biologically active form of the peptide and that a principle role of Ser6 is to favor the proportion in the trans conformation.…”