Carbon-13 nuclear-magnetic-resonance studies on selected amino acids, peptides, and proteins.

Abstract

Natural abundance high‐resolution carbon‐13 Fourier transform nuclear magnetic resonance (13C NMR) studies at 25 MHz have been carried out on selected amino acids, linear peptides and proteins. The pH dependence of the 13C resonances has been studied in detail in several cases. For l‐histidine, the 13C chemical shifts of all six carbon atoms are sensitive to the ionization state of the titrating carboxyl, imidazole and amino groups. The data for each carbon resonance have been computer‐fitted to a sum of three theoretical curves based on a simple proton‐association equilibrium for each transition. The extent of chemical shift change upon ionization of a nearby group has been interpreted in terms of two competing effects, inductive (through‐bond) and electric field (through‐space) effects.The 13C NMR spectra of the amino‐terminal 1–13, 1–15, and 1–20 peptides of bovine pancreatic ribonuclease A have been analyzed. Detailed resonance assignments have been made based on comparisons with the results for the free amino acids, shift effects for amino acids in small peptides, internal peptide bond effects and differences in amino acid composition. There is considerable correspondence between the observed 13C chemical shifts of these peptides and those predicted from the shifts of the component amino acids.Proteins of varying molecular weight have been studied to evaluate the potential of 13C NMR investigations of structure and conformation. A comparison has been made between the 13C NMR spectra of hen egg‐white lysozyme in its native and reduced states. The spectrum of the fully reduced species appears to be well represented by the sum of the spectra of the individual amino acids, both in chemical shift and line width, whereas that for the native form exhibits consistently broader resonances. Similar results were obtained for ribonuclease A. At the current level of resolution, it is impossible to discern whether this line broadening derives from shorter relaxation time effects due to restricted motion, chemical shift non‐equivalence between the same chemical groups, or both. However, it does appear that there are separately resolved peaks corresponding to Cβ‐carbons for different threonyl residues, as well as for the C‐4 ring carbons of the several tyrosyl residues in native hen egg‐white lysozyme. Spectra of carbonic anhydrases (Mr∼ 30000), which lack disulphide bonds, showed somewhat poorer resolution than the smaller proteins.