Residue-specific millisecond to microsecond fluctuations in bacteriorhodopsin induced by disrupted or disorganized two-dimensional crystalline lattice, through modified lipid-helix and helix-helix interactions, as revealed by 13C NMR.
We have recorded 13C NMR spectra of [3-13C]-, [1-13C]Ala-, and [1-13C]Val-labeled bacteriorhodopsin (bR), W80L and W12L mutants and bacterio-opsin (bO) from retinal-deficient E1001 strain, in order to examine the possibility of their millisecond to microsecond local fluctuations with correlation time in the order of 10(-4) to 10(-5) s, induced or prevented by disruption or assembly of two-dimensional (2D) crystalline lattice, respectively, at ambient temperature. The presence of disrupted or disorganized 2D lattice for W12L, W80L and bO from E1001 strain was readily visualized by increased relative proportions of surrounding lipids per protein, together with their broadened 13C NMR signals of transmembrane alpha-helices and loops in [3-13C]Ala-labeled proteins, with reference to those of wild-type. In contrast, 13C CP-MAS NMR spectra of [1-13C]Ala- and Val-labeled these mutants were almost completely suppressed, owing to the presence of fluctuations with time scale of 10(-4) s interfered with magic angle spinning. In particular, 13C NMR signals of [1-13C]Ala-labeled transmembrane alpha-helices of wild-type were almost completely suppressed at the interface between the surface and inner part (up to 8.7 A deep from the surface) with reference to those of the similarly suppressed peaks by Mn(2+)-induced accelerated spin-spin relaxation rate. Such fluctuation-induced suppression of 13C NMR peaks from the interfacial regions, however, was less significant for [1-13C]Val-labeled proteins, because fluctuation motions in Val residues with bulky side-chains at the C(alpha) moiety were modified to those of longer correlation time (>10(-4) s), if any, by residue-specific manner. To support this view, we found that such suppressed 13C NMR signals of [1-13C]Ala-labeled peaks in the wild-type were recovered for D85N and bO in which correlation times of fluctuations were shifted to the order of 10(-5) s due to modified helix-helix interactions as previously pointed out [Biochemistry, 39 (2000) 14472; J. Biochem. (Tokyo) 127 (2000) 861].
Saitô
Hazime
H
Department of Life Science, Graduate School of Science, Himeji Institute of Technology, Harima Science Garden City, Kouto 3 chome, Kamigori, Hyogo 678-1297, Japan. saito@sci.himeji-tech.ac.jp
Tsuchida
Takahiro
T
Ogawa
Keizi
K
Arakawa
Tadashi
T
Yamaguchi
Satoru
S
Tuzi
Satoru
S
D001436:Bacteriorhodopsins / Q000737:chemistry* / Q000235:genetics; D002247:Carbon Isotopes; D003460:Crystallization; D019617:Halobacterium salinarum / Q000737:chemistry* / Q000235:genetics; D008563:Membrane Lipids / Q000737:chemistry; D008565:Membrane Proteins / Q000737:chemistry; D009154:Mutation; D019906:Nuclear Magnetic Resonance, Biomolecular / Q000379:methods; D017510:Protein Folding; D017433:Protein Structure, Secondary; D013696:Temperature; D013997:Time Factors
D002247:Carbon Isotopes; D008563:Membrane Lipids; D008565:Membrane Proteins; D001436:Bacteriorhodopsins