Dynamic aspect of bacteriorhodopsin as a typical membrane protein as revealed by site-directed solid-state 13C NMR.
We demonstrate here a general feature of dynamic aspect of membrane proteins as revealed by site-directed 13C NMR studies on bacteriorhodopsin (bR) as a typical membrane protein and a variety of mutants at ambient temperature. 13C NMR signals of [3-13C]Ala- or [1-13C]Val-labeled proteins were assigned regio-specifically with reference to the data of the conformation-dependent 13C chemical shifts from model polypeptides, followed by site-specific assignment based on site-directed mutants. Revealed picture of membrane protein at ambient temperature is not static in contrast to anticipation from crystalline structures but flexible enough to undergo a variety of local fluctuations with frequencies from 10(2) to 10(8)Hz, as pointed out already. This picture was further refined by taking into account of residue-specific dynamics of interfacial domains between the surface and inner part of the transmembrane helices and conformational fluctuation induced by the presence of a kinked structure. The residue-specific dynamics of the former was revealed by observation of broadened or suppressed peaks from the interfacial domains caused by acquisition of internal fluctuation motions interfered with frequencies of proton decoupling or magic angle spinning. The presence of such suppressed peaks due to molecular fluctuations in the interfacial domains was further confirmed by insensitivity of the peak-intensities from the interfacial domains in spite of the presence of accelerated relaxation rate to nearby residues from surface bound Mn2+ ion. Further, conformational change of the transmembrane alpha-helix F due to a plausible kinked structure at Pro 186 was confirmed in view of specific displacements of Ala 184 and Val 187 13C NMR peaks from chemically synthesized [3-13C]Ala(184)-, [1-13C]Val(187)-labeled wild type and P186L mutant of transmembrane fragment F(164-194) incorporated into lipid bilayer. It is emphasized that the observed displacement of [3-13C]-labeled Ala 184 peak at 17.4 ppm in the presence of kinked structure in this model peptide is consistent with that of intact protein at 17.27 ppm.
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
Yamaguchi
Satoru
S
Okuda
Hideyasu
H
Shiraishi
Aya
A
Tuzi
Satoru
S
D000596:Amino Acids / Q000737:chemistry*; D001436:Bacteriorhodopsins / Q000737:chemistry*; D002247:Carbon Isotopes; D008051:Lipid Bilayers / Q000737:chemistry*; D009682:Magnetic Resonance Spectroscopy / Q000379:methods*; D008560:Membrane Fluidity; D008565:Membrane Proteins / Q000737:chemistry; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D011487:Protein Conformation; D017433:Protein Structure, Secondary; D017434:Protein Structure, Tertiary; D013329:Structure-Activity Relationship; D013499:Surface Properties; D013696:Temperature
D000596:Amino Acids; D002247:Carbon Isotopes; D008051:Lipid Bilayers; D008565:Membrane Proteins; D001436:Bacteriorhodopsins