Conformation and dynamics changes of bacteriorhodopsin and its D85N mutant in the absence of 2D crystalline lattice as revealed by site-directed 13C NMR.
13C NMR spectra of [3-(13)C]Ala- and [1-(13)C]Val-labeled D85N mutant of bacteriorhodopsin (bR) reconstituted in egg PC or DMPC bilayers were recorded to gain insight into their secondary structures and dynamics. They were substantially suppressed as compared with those of 2D crystals, especially at the loops and several transmembrane alphaII-helices. Surprisingly, the 13C NMR spectra of [3-(13)C]Ala-D85N turned out to be very similar to those of [3-(13)C]Ala-bR in lipid bilayers, in spite of the presence of globular conformational and dynamics changes in the former as found from 2D crystalline preparations. No further spectral change was also noted between the ground (pH 7) and M-like state (pH 10) as far as D85N in lipid bilayers was examined, in spite of their distinct changes in the 2D crystalline state. This is mainly caused by that the resulting 13C NMR peaks which are sensitive to conformation and dynamics changes in the loops and several transmembrane alphaII-helices of the M-like state are suppressed already by fluctuation motions in the order of 10(4)-10(5) Hz interfered with frequencies of magic angle spinning or proton decoupling. However, 13C NMR signal from the cytoplasmic alpha-helix protruding from the membrane surface is not strongly influenced by 2D crystal or monomer. Deceptively simplified carbonyl 13C NMR signals of the loop and transmembrane alpha-helices followed by Pro residues in [1-(13)C]Val-labeled bR and D85N in 2D crystal are split into two peaks for reconstituted preparations in the absence of 2D crystalline lattice. Fortunately, 13C NMR spectral feature of reconstituted [1-(13)C]Val and [3-(13)C]Ala-labeled bR and D85N was recovered to yield characteristic feature of 2D crystalline form in gel-forming lipids achieved at lowered temperatures.
Department of Life Science, Himeji Institute of Technology, University of Hyogo, Harima Science Garden City 678-1297, Japan.
Kazutoshi
K
Yamamoto
Satoru
S
Tuzi
Hazime
H
Saitô
Izuru
I
Kawamura
Akira
A
Naito
D019943:Amino Acid Substitution; D001436:Bacteriorhodopsins / Q000737:chemistry* / Q000235:genetics*; D003460:Crystallization; D004134:Dimyristoylphosphatidylcholine / Q000737:chemistry; D019617:Halobacterium salinarum / Q000737:chemistry / Q000235:genetics; D008051:Lipid Bilayers / Q000737:chemistry; D016297:Mutagenesis, Site-Directed; D019906:Nuclear Magnetic Resonance, Biomolecular; D010713:Phosphatidylcholines / Q000737:chemistry; D011487:Protein Conformation; D013816:Thermodynamics
D008051:Lipid Bilayers; D010713:Phosphatidylcholines; D001436:Bacteriorhodopsins; D004134:Dimyristoylphosphatidylcholine