Multinuclear two‐dimensional NMR: Assignments of natural abundance polypeptide 13C, 1H and 15N chemical shifts and demonstration of isomer interconversion

Abstract

AbstractThe polypeptide carbobenzoxy‐glycyl‐L‐prolyl‐L‐leucyl‐L‐alanyl‐L‐proline (0.2 M in DMSO‐d6) was investigated using 13C, 1H and 15N NMR in natural abundance at 4.7 tesla. The existence of cis–trans‐Gly‐Pro and ‐Ala‐Pro bonds permits up to four isomers, and all four were observed (in a 60:30:7:3 ratio). 13C shifts of the proline β‐CH2 resonances are consistent only with the 60% form being trans–trans. The 30% form is either trans–cis or cis–trans (order as above) and was tentatively assigned as cis‐trans on the basis of relaxation behavior. Refocused INEPT studies aided the 13C assignments. The 15N data were obtained using both NOE and INEPT excitation, with signals evident for the three major isomers. The spectra were analysed by starting from the 13C data, which were assigned based on known regularities in peptide spectra. A 13C1H heteronuclear two‐dimensional chemical shift correlation experiment allowed direct assignment of proton shifts for major and minor isomers. The NH proton shifts were assigned by running a homonuclear two‐dimensional chemical shift correlation experiment and noting the correlation with the previously assigned α‐CH protons. The 15N resonances were then assigned from a 15N1H heteronuclear two‐dimensional chemical shift correlation experiment, relating the 15N signals directly to the NH proton resonances. Isomer interconversion between the two major isomers was demonstrated by performing a magnetization transfer homonuclear 2D experiment. Off‐diagonal intensity was noted relating the major and minor isomer alanine NH proton, as well as for the major and minor isomer leucine NH protons.