Two-dimensional NMR approaches to structure determination of small molecules in solution and bound to proteins.

摘要

Nuclear magnetic resonance (NMR) spectroscopy is a powerful approach to characterize molecules and elucidate their structures. Here, two-dimensional (2D) NMR spectroscopy was used to determine the structures of four types of molecules: (1) UAB retinoids in organic solution; (2) The tripeptide, Ac-Pro-Gly-Pro-OH, in aqueous and organic solutions; (3) Cytidine 3 ′-monophosphate (3CMP) bound to ribonuclease A (RNase A); and (4) nicotinic acid adenine dinucleotide (NaAD) or nicotinamide adenosine dinucleotide (NAD+) bound to NAD synthetase.; In the first article, the structures of 22 different retinoid analogs, which were synthesized by others, were determined. To realize the structure of each retinoid, 1D 1H and 13C NMR spectral assignments were first obtained for each of these retinoids by using 2D homonuclear and heteronuclear correlated spectroscopy. After 1H and 13C assignments, the correct E/Z stereochemical assignments were made by using the nuclear Overhauser effect (NOE). By this method, 9 Z, 13Z and all-E isomers of three important UAB retinoids were determined.; In the second article, the structures of four isomers of the chemoattractant tripeptide were determined. Four different conformations of the tripeptide were identified with either cis or trans conformations about the Ac-Pro and Gly-Pro amide bonds. These isomers are named the trans-trans, cis-trans, trans-cis, and cis-cis isomers. 2D heteronuclear spectroscopies such as heteronuclear multiple quantum coherence (HMQC), heteronuclear relay spectroscopy (HMQC-TOCSY), heteronuclear correlation spectroscopy (HETCOR) and heteronuclear multiple bond correlation (HMBC) were used to assign the 1H and 13C chemical shifts of each isomer of Ac-Pro-Gly-Pro-OH.; In the third article, the conformation of a mononucleotide, 3 ′CMP, bound to RNase A was determined. 2D transferred NOESY (TrNOESY) was used to determine the conformation of this small ligand bound to a small protein.; In the last article, the bound conformation of dinucleotides, NaAD or NAD+, were determined to NAD synthetase. NAD synthetase catalyzes the last step in NAD+ biosynthesis, transforming NaAD into NAD+ by a two-step reaction. To understand the catalytic mechanism of the enzyme and design new transition-state inhibitors of the enzyme, the conformations of enzyme-bound NaAD and NAD+ were determined and compared to the X-ray structure of the intermediate NAD-adenylate complex with the protein. (Abstract shortened by UMI.)