A high‐resolution solid‐state 13 C‐NMR study on [1‐ 13 C]Ala and [3‐ 13 C]Ala and [1‐ 13 C]Leu and Val‐labelled bacteriorhodopsin

We have recorded 100.7‐MHz high‐resolution solid‐state 13 C‐NMR spectra of [3‐ 13 C]Ala, [1‐ 13 C]Ala‐labelled, Leu‐labelled and Val‐labelled bacteriorhodopsin (bR), to analyze the conformation and dynamics of transmembrane α helices and hydration‐induced conformational changes. We assigned the 13 C‐NMR signals of these 13 C‐labelled amino acid residues to portions of the transmembrane α helices, loops and N‐terminus and C‐terminus, based on the conformation‐dependent 13 C chemical shift. The assignment of peaks to the transmembrane α helices is straightforward in view of the characteristic 13 C chemical shifts of the Cβ and carbonyl carbons, referred to the data of the model system. The signals of the transmembrane α helices were further divided into three or four peaks which are ascribed to either a dispersion of torsion angles of the α helices or variation of environments around the helices. In addition, we found that conformation of the N‐terminus and C‐terminus of bR is virtually the random‐coil form which undergoes rapid reorientational motion in the hydrated system. Further, we show that the higher‐order (secondary and/or tertiary) structure of bR is influenced by the hydration/dehydration process of the purple membrane, as viewed from the peak profile of the Ala Cβ 13 C signals. This change of structure occurs between relative humidities of 2% and 4%, consistent with the shift of the absorption maxima of retinal, which arise from the protonation/deprotonation processes of the Schiff base. In contrast, no such change of the carbonyl signals occurs for the Leu, Val and Ala residues. For the latter two residues, some spectral changes were noted at the stage of full hydration.