Temperature‐Dependent Conformational Change of Bacteriorhodopsin as Studied by Solid‐State ¹³C NMR

Abstract

Cross-polarization and dipolar-decoupled magic-angle spinning "C-NMR spectra of [3-'TC]Ala-labelled bacteriorhodopsin were obtained for hydrated purple membrane in the temperatures range 23 "C to -1 10°C. Well-resolved "C-NMR signals were observed either at ambient temperature or at -20°C but were broadened considerably at lower temperature below -40°C. This situation was interpreted in terms of the presence of exchange processes with a rate constant of 10' s-' at ambient temperature among several conformations slightly different from each other. We found that such an exchange process was strongly influenced by the manner of organization of the lipid bilayers depending upon the presence or absence of cations responsible for electric shielding of negative charge at the polar head groups. The manner of organization of the lipid bilayers was conveniently characterized by a characteristic temperature at which the methyl peaks of fatty acyl groups of lipids in the purple membrane were suppressed due to interference of motional frequency with the decoupling frequency (1 0-100 kHz) for preparations containing 10 mM NaCl or CaCl,. No such spectral change in the absence of these cations was noted even if a preparation was cooled to -1 10 "C. The secondary structures of [3-'3C]Ala-labelled bacteriorhodopsin was not always identical at temperatures between ambient and low temperatures, since the "C chemical shifts and relative peak intensities for purple membrane preparations containing these salts changed with temperature in the range -110°C to 23°C. In particular, we found that some residues involving Ala residues at the a,,-helix and loop region were converted at temperatures below -60°C to a conformation involving a,-helix. In other words, some portion of the a-helical conformation of bacteriorhodopsin proposed from results obtained by cryo-electron microscopy, at very low temperatures, is not always retained at ambient temperature.

15N T2′ relaxation times of bacteriorhodopsin transmembrane amide nitrogens

“…Since one can expect a reduction in temperature to reduce motion frequencies, this indicates that the internal dynamics of BR's helices are on the lowfrequency side of this millisecond time-scale. This hypothesis is consistent with the result obtained by Tuzi et al 50 from CP/MAS experiments on specifically labelled [3-13 C]Ala-PM. They observed that the 13 C NMR spectral resolution in the alanine methyl carbons region deteriorates substantially at temperatures lower than 40°C owing to more efficient T 2 0 relaxation.…”

Site‐directed 13C solid‐state NMR studies on membrane proteins: strategy and goals toward revealing conformation and dynamics as illustrated for bacteriorhodopsin labeled with [1‐13C]amino acid residues

“…12(B), right]. 10,24 Therefore, the spectral pattern shown in Fig. 12(C) can be well recognized as a 2D array, although the ionic strength of the 10 mM NaCl used for this experiment is low as compared with the previous experiments.…”

“…The resulting 13 C NMR signals were well resolved to yield 12 and 9 signals for [3-13 C]Ala-and [1- 13 C]Val-bR of fully hydrated preparations, respectively, at ambient temperature, and consequently many such resolved peaks were assigned initially in a regiospecific manner based on the conformationdependent displacements of 13 C NMR signals, 7 -9 and subsequently in a site-directed manner in view of reduced peak intensities of site-directed mutants with reference to the peak of the wild type. In such a case, the 13 C NMR signals are almost fully visible 10 as far as [3-13 C]Ala-bR from a purple membrane consisting of 2D crystals of hexagonally packed trimeric forms 11 is concerned. In this way, we accomplished the assignment of the 13 C NMR signals (over 60%) from [3-13 C]Ala-bR and obtained much invaluable information about the conformation and dynamics of bR of biological significance under physiological conditions, 2,12 -25 as summarized in Table 1.…”

Dynamic aspect of bacteriorhodopsin as a typical membrane protein as revealed by site-directed solid-state 13C NMR

“…It is emphasized that the presence of such low frequency motions at the interfacial domains depends upon the type of amino-acid residues of less bulky substituent at C a such as Ala and Gly, with the correlation time in the order of 10 À4 s as same as that of the loop regions, although this time scale is shifted to longer one as viewed from the 13 C NMR signals of Val, Ile and Trp residues with bulky side-chains. In contrast, the correlation time for motions of the transmembrane a-helices is in the order of 10 À2 s for purple membrane in which bR is hexagonally packed [35] but changed to the order of 10 À4 s in the case of monomeric state [14]. It is emphasized that 13 C NMR signals of more flexible portions such as C-or N-terminal residues can be detected by DD-MAS experiment alone, because dipolar interactions for such domains essential for CP-MAS experiment are time-averaged to zero.…”