Conformation and dynamics of membrane proteins and biologically active peptides as studied by high-resolution solid-state 13C NMR

Abstract

We have explored an empirical approach to clarify the three-dimensional structure and dynamics of membrane proteins such as bacteriorhodopsin (bR) based on 13C NMR measurements, utilizing the concept of the conformation-dependent displacements of 13C chemical shifts as determined by cross polarization-magic angle spinning (CP-MAS) and dipolar decoupled-magic angle spinning (DD-MAS) methods. This is possible because 13C chemical shifts of the amino acid residues under consideration are appreciably displaced, up to 8 ppm, depending upon particular conformations from several portions of membrane proteins such as the transmembrane α-helices, loops, N- or C-terminus, etc. as referred to the data accumulated to date of an appropriate model system. It is also possible to distinguish a region characterized by a variety of backbone motions with at least three different time scales from NMR data: rapid motions with correlation times shorter than 10 −8 s, intermediate motions with correlation times of 10 −4 to 10 −5 s, and slow motions with a time scale of 10 −2 s. In addition, we also explored a non-empirical approach to reveal the three-dimensional structure of a smaller molecular system such as biologically active peptides as a messenger molecule in signal transduction, based on accurately determined appropriate sets of interatomic distances as determined by rotational echo double resonance (REDOR). Examination of 13C or 15N chemical shifts before and after REDOR experiments proved to be an indispensable means to examine whether or not conformations of several kinds of 13C, 15N-doubly labeled samples at different positions are not changed all the time. Here, we summarize some illustrative examples to this end, selected from our recent studies on [3- 13C]Ala-labeled bR and biologically active peptides such as enkephalins.