1996 Fiscal Year Final Research Report Summary
Three-dimensional Structure and Dynamics of Membrane Proteins by NMR
| Project/Area Number |
06454666
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| Research Category |
Grant-in-Aid for Scientific Research (B)
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| Allocation Type | Single-year Grants |
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| Section | 一般 |
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| Research Field |
Biophysics
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| Research Institution | Himeji Institute of Technology |
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Principal Investigator |
SAITO Hazime Himeji Institute of Technol, Fac. Science, professor, 理学部, 教授 (30100150)
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| Co-Investigator(Kenkyū-buntansha) |
TUZI Satoru Himeji Inst. Technol, Instructer, 理学部, 助手 (60227387)
NAITO Akira Himeji Inst. Technol. Fac. Sci. Assoc. Professor, 理学部, 助教授 (80172245)
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| Project Period (FY) |
1994 – 1996
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| Keywords | Membrane Proteins / Solid-state NMR / Three-dimensional Structure / Bacteriorhodopsin / Dynamics |
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| Research Abstract |
We have analyzed conformation and dynamics of [3-^<13>]Ala-bacteriorhodopsin by high-resolution solid-state NMR and obtained the following results.
(1) Regio-and site-specific assignments of peaks : We have assigned the observed ^<13>C signals to trnsmembrane helices, loops and N-or C-terminus on the basis of the conformation-dependent displacements of peaks. Further, we have completed 50% of site-specific assignments the peaks to the respective site of amino-acid residues on the basis of comparison of the spectra between wild type and mutant strains, spectral changes either by proteolytic digestion or pH titration, comparison of ^<13>C NMR spectra of chemically synthesized fragments, etc. We further pointed out the presence of the alpha-helices in the C-terminus protruding from the membrane surface.
(2) Temperature dependent conformational change : We found substantial line-broadening in the signals recorded at temperatures lower than -20ﾟC due to irregular freezing of molecular chains undergoing fractuation at ambient temperature with rate constant larger than 10^2 S-^<-1>. This is mainly causedd by modulation of lipid-packing caused by electrostatic interaction between cation and lipids
(3) Interactions with retinal, lipids and detrgents : It was found that substantial conformational change due to protein-retinal interaction was associated with bacterio-opsin in which retinal was removed. It is also interesting to note that molecular motions of intermediate time scale as long as 10^<-4>-10^<-5> s were induced at the loop region by removal of retinal. In addition, de-lipidation or solubilization by detergenst resulted in significant conformational changes.
(4) Backbone dynamics : We denoted the presence of rapid motion in the randomly coiled C-terminus region in which CP-MAS signals were suppressed, in addition to slow and intermediate motions mentioned above.
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