Significance of low-frequency local fluctuation motions in the transmembrane B and C alpha-helices of bacteriorhodopsin, to facilitate efficient proton uptake from the cytoplasmic surface, as revealed by site-directed solid-state 13C NMR.
13C NMR spectra of [1-13C]Val- or -Pro-labeled bacteriorhodopsin (bR) and its single or double mutants, including D85N, were recorded at various pH values to reveal conformation and dynamics changes in the transmembrane alpha-helices, in relation to proton release and uptake between bR and the M-like state caused by modified charged states at Asp85 and the Schiff base (SB). It was found that the D85N mutant acquired local fluctuation motion with a frequency of 10(4) Hz in the transmembrane B alpha-helix, concomitant with deprotonation of SB in the M-like state at pH 10, as manifested from a suppressed 13C NMR signal of the [1-13C]-labeled Val49 residue. Nevertheless, local dynamics at Pro50 neighboring with Val49 turned out to be unchanged, irrespective of the charged state of SB as viewed from the 13C NMR of [1-13C]-labeled Pro50. This means that the transmembrane B alpha-helix is able to acquire the fluctuation motion with a frequency of 10(4) Hz beyond the kink at Pro50 in the cytoplasmic side. Concomitantly, fluctuation motion at the C helix with frequency in the order of 10(4) Hz was found to be prominent, due to deprotonation of SB at pH 10, as viewed from the 13C NMR signal of Pro91. Accordingly, we have proposed here a novel mechanism as to proton uptake and transport based on a dynamic aspect that a transient environmental change from a hydrophobic to hydrophilic nature at Asp96 and SB is responsible for the reduced p Ka value which makes proton uptake efficient, as a result of acquisition of the fluctuation motion at the cytoplasmic side of the transmembrane B and C alpha-helices in the M-like state. Further, it is demonstrated that the presence of a van der Waals contact of Val49 with Lys216 at the SB is essential to trigger this sort of dynamic change, as revealed from the 13C NMR data of the D85N/V49A mutant.
Kira
Atsushi
A
Department of Life Science, Graduate School of Science, Himeji Institute of Technology, Harima Science Garden City, Kamigori, 678-1297 Hyogo, Japan.
Tanio
Michikazu
M
Tuzi
Satoru
S
Saitô
Hazime
H
D001436:Bacteriorhodopsins / Q000737:chemistry* / Q000235:genetics / Q000528:radiation effects*; D002247:Carbon Isotopes; D002462:Cell Membrane / Q000737:chemistry*; D003593:Cytoplasm / Q000737:chemistry*; D008027:Light; D018499:Linear Energy Transfer / Q000528:radiation effects; D009682:Magnetic Resonance Spectroscopy / Q000379:methods*; D009038:Motion; D016297:Mutagenesis, Site-Directed; D011487:Protein Conformation / Q000528:radiation effects; D017434:Protein Structure, Tertiary / Q000528:radiation effects; D017494:Proton Pumps / Q000737:chemistry* / Q000528:radiation effects; D011522:Protons; D011994:Recombinant Proteins / Q000737:chemistry / Q000528:radiation effects; D013329:Structure-Activity Relationship
D002247:Carbon Isotopes; D017494:Proton Pumps; D011522:Protons; D011994:Recombinant Proteins; D001436:Bacteriorhodopsins