Abstract
Abstract Pentapeptides containing either glycine, l-alanine, l-valine, l-leucine, or l-isoleucine as central residue in the pattern glycylglycyl-X-glycylglycine have been studied by proton-decoupled natural abundance 13C Fourier transform nuclear magnetic resonance spectroscopy. Resonances were assigned by comparison with the free amino acids and by detailed observation of changes in chemical shifts with pH over the range 1.32 to 10.43. The effect of varying the central residue on the chemical shifts of the flanking glycine residues was established. Small effects ascribable to incipient aggregation were noted, both in terms of chemical shifts and relaxation properties. Each resonance was categorized according to the degree of sensitivity to the state of protonation of the terminal groups, and the pK values at 26° for those groups were determined from the pH dependence of the most sensitive resonances. The sensitive terminal resonances undergo broadening which is maximum near the pK. Spin-lattice relaxation times, T1, of protonated carbon nuclei were measured at two or more pH values in all cases except for the glycine pentapeptide. For each peptide Cα resonances showed gradients in increasing T1 to each side from the central residue, indicative of contributions from segmental motion and internal rotation along the backbone in addition to the over-all rotational motion of the molecule. Internal rotational modes are expressed as well in the aliphatic side chains, especially by the most peripheral methyl groups for which spinning around the attaching bond is clearly prominent. Comparison of the anionic and cationic forms of the alanine, valine, and leucine peptides indicates that the rate of internal rotation of Cα of the NH2-terminal glycine residues increases with deprotonation of the ammonium group.
Highlights
Valine, L-leucine, or L-isoleucine as central residue in the pattern glycylglycyl-X-glycylglycine have been studied by proton-decoupled natural abundance W Fourier transform nuclear magnetic resonance spectroscopy
The pentapeptides were synthesized by the Merrifield solid phase technique [32] as outlined by Stewart and Young [33]
3 Because of instrumental limitations the nuclear Overhauser effect (NOE) and Tr measurements were made at different frequencies. mechanism involved is frequency dependent
Summary
Peptide Synthesis-The amino acids L-alanine, L-valine, L-isoleucine, and L-leucine (Pierce Chemical Company) were converted to the respective t-1308 derivatives according to the method of Schnabel [30] by treatment with t-butylazidoformate (Pierce).This procedure was employed in the conversion of glycylglycine (Pierce) to the t-BOC form by reaction in dioxane-Hz0(1: 1, v/v) at pH 9.5 for 9 hours. Peptide Synthesis-The amino acids L-alanine, L-valine, L-isoleucine, and L-leucine (Pierce Chemical Company) were converted to the respective t-1308 derivatives according to the method of Schnabel [30] by treatment with t-butylazidoformate (Pierce). This procedure was employed in the conversion of glycylglycine (Pierce) to the t-BOC form by reaction in dioxane-Hz0. (1: 1, v/v) at pH 9.5 for 9 hours. A three st,age procedure was employed whereby t-BOC-glycylglycine was initially coupled to the chloromethylated resin (Bio-Beads S-X2,200 to 400 mesh, Bio-Rad) by reaction with triethylamine in absolute ethanol for 30 hours.
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