¹³C NMR studies of insulin. Part I—Spectral assignments

Abstract

Natural abundance 75 MHz I3C NMR spectral assignments are reported for bovine and porcine zinc insulin in solution. A large number of protein resonances are well resolved, and approximately 80% of these have been assigned to either residue types or to specific sites within the protein. Assignment techniques included consideration of free amino acid or peptide shifts, pH studies and comparison of sequence and spectral differences between bovine and porcine insulin, in addition to the use of NMR relaxation times. The DEPT spectral editing technique was also found to be particularly valuable as an assignment aid. This technique allows subspectra containing only CH, CH, or CH, carbon types to be generated. The method also produces signal enhancement relative to broad band decoupled I3C NMR spectra of large proteins which generally have reduced nuclear Overhauser enhancements.

Proton NMR studies of manganese ion binding to tRNAderived acceptor arm duplexes

“…within a radius of about 1 nm) it accelerates the relaxation of the proton spin drastically, giving rise to a broadened NMR line. The extent of the linewidth increase varies as r-6 (r-distance between proton and paramagnetic ion) [7]. From the fact that there is only one uniform set of spectra with certain specifically broadened peaks it must be concluded that there is a rapid exchange (on the NMR timescale) between bound and free Mn2+ ions.…”

“…The 5' and 2' OH groups of ribose were protected by 4,4'-dimethoxytrityl and tert-butyldimethylsilyl protecting groups, respectively. Protection of the exocyclic amines in adenine and guanine was achieved by dimethylaminomethylene protection groups [7,8]. The purification of the oligonucleotides was performed by HPLC on a Vydac C4 column.…”

“…Thus it appeared interesting to address this question by studying various acceptor stem helices derived from different tRNAs with respect to their Mg2+ binding behaviour by means of proton NMR spectroscopy using Mn2+ as a probe since it is wellestablished that manganese can substitute for magnesium in many biological systems without significantly hampering their function [7]. A similar approach using paramagnetic ions that permitted detection of divalent metal ion binding sites in whole tRNA molecules was used, e.g., already by Chao and Kearns [8] and Hurd et al [9].…”

The 3'-terminal end (NCCA) of tRNA determines the structure and stability of the aminoacyl acceptor stem.

“…The resonances of protons in the vicinity of paramagnetic probes were broadened with the excess linewidth varying as r-6, where r is the distance between ion and proton (11). Hence, there is a preferred binding site for Mn2+ and, correspondingly, for Mg2+ in the vicinity ofbp 2 and 3.…”

“…These were different in magnitude and sign for the individual base pairs for the duplexes with and without an ACCA end. To check whether these small differences in chemical shift of NH resonances are related to variations in divalent ion binding affinity along (11,12). The imino resonance regions of the proton NMR spectra of the 14-mer and the 18-mer before and after addition of MnCl2, respectively, are presented in Fig.…”