Abstract

Solid state NMR experiments will be employed to investigate three membrane proteins -- Bacteriohodopsin (bR), Rhodopsin (RHO), and Halorhodopsin (hR). The bR investigations will focus on (1) improving our understanding of the Opsin Shift in bR through optical and NMR studies of 6-s-trans model compounds; (2) elucidation of the proton translocation mechanism through 13C-15N rotational echo double resonance (REDOR) and 13C-13C rotational resonance (R2) determinations of the Lys-216 sidechain conformation; (3) high resolution 1H NMR studies designed to determine the existence and structure of the hydrogen bonded chain; (4) low temperature trapping and detection of the protonation/deprotonation of amino acid sidechains such as Tyr and Asp in bR photointermediates; (5) 19F-19F and 13C-13C rotational resonance experiments designed to determine the location of Trp's and Tyr in the retinal binding pocket. The hR experiments will be aimed at studying the opsin shift mechanism in this protein, the state of the two His residues, and the binding of anions. Finally, the RHO experiments will focus on chromophore structure in bathorhodopsin and will rely on comparison of chemical shifts between RHO, isorhodopsin, and batho. Large perturbations in the shifts will be investigated further with R2 structural studies. In particular, R2 lineshapes will be employed to search for twist about single (and perhaps double) bonds in the photo excited chromophore.

  Funding Agency

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
5R01GM023289-18
Application #
3271579
Study Section
Molecular and Cellular Biophysics Study Section (BBCA)
Project Start
1976-05-01
Project End
1995-06-30
Budget Start
1993-07-01
Budget End
1994-06-30
Support Year
18
Fiscal Year
1993
Total Cost
Indirect Cost

  Institution

Name
Massachusetts Institute of Technology
Department
Type
Organized Research Units
DUNS #
City
Cambridge
State
MA
Country
United States
Zip Code
02139

  Publications

Petkova, Aneta T; Baldus, Marc; Belenky, Marina et al. (2003) Backbone and side chain assignment strategies for multiply labeled membrane peptides and proteins in the solid state. J Magn Reson 160:1-12
Lansing, Jonathan C; Hu, Jingui G; Belenky, Marina et al. (2003) Solid-state NMR investigation of the buried X-proline peptide bonds of bacteriorhodopsin. Biochemistry 42:3586-93
Petkova, Aneta T; Hatanaka, Minoru; Jaroniec, Christopher P et al. (2002) Tryptophan interactions in bacteriorhodopsin: a heteronuclear solid-state NMR study. Biochemistry 41:2429-37
Hatcher, Mary E; Hu, Jingui G; Belenky, Marina et al. (2002) Control of the pump cycle in bacteriorhodopsin: mechanisms elucidated by solid-state NMR of the D85N mutant. Biophys J 82:1017-29
Lansing, Jonathan C; Hohwy, Morten; Jaroniec, Christopher P et al. (2002) Chromophore distortions in the bacteriorhodopsin photocycle: evolution of the H-C14-C15-H dihedral angle measured by solid-state NMR. Biochemistry 41:431-8
Jaroniec, Christopher P; Filip, Claudiu; Griffin, Robert G (2002) 3D TEDOR NMR experiments for the simultaneous measurement of multiple carbon-nitrogen distances in uniformly (13)C,(15)N-labeled solids. J Am Chem Soc 124:10728-42
Jaroniec, C P; Tounge, B A; Herzfeld, J et al. (2001) Frequency selective heteronuclear dipolar recoupling in rotating solids: accurate (13)C-(15)N distance measurements in uniformly (13)C,(15)N-labeled peptides. J Am Chem Soc 123:3507-19
Jaroniec, C P; Lansing, J C; Tounge, B A et al. (2001) Measurement of dipolar couplings in a uniformly (13)C,(15)N-labeled membrane protein: distances between the Schiff base and aspartic acids in the active site of bacteriorhodopsin. J Am Chem Soc 123:12929-30
Griffiths, J M; Bennett, A E; Engelhard, M et al. (2000) Structural investigation of the active site in bacteriorhodopsin: geometric constraints on the roles of Asp-85 and Asp-212 in the proton-pumping mechanism from solid state NMR. Biochemistry 39:362-71
Jaroniec, C P; Tounge, B A; Rienstra, C M et al. (2000) Recoupling of heteronuclear dipolar interactions with rotational-echo double-resonance at high magic-angle spinning frequencies. J Magn Reson 146:132-9

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