Abstract

High resolution, solid state NMR experiments will be used to investigate three membrane proteins - rhodopsin(RHOD), bacteriorhodopsin(BR), and halorhodopsin(HR). RHOD will be prepared with a variety of 13C labelled retinals and several aspects of the retinal configuration will be studied. The BR investigations will involve 13C labelled retinals and 13C, 15N, and 17O labelled amino acids. The 15N-1H bond distance in the Schiff base will be measured with DIPSHIFT experiments. The configuration of retinal in photointermediates, like M412, will be determined and the involvement of specific amino acids in the BR proton wire will be investigated by looking for deprotonation effects in 13C and 17O spectra. Resonances will be assigned with a new approach involving 15N and doubly 13C labelled amino acids together with heteronuclear chemical shift correlation and spin diffusion experiments (SDE). SDE will also be used to establish the proximity of retinal to particular amino acids by studying BR in which both retinal and the sidechains are labelled. The dynamic structure of BR will be studied with 13C and 2H NMR and the structure of the protein will be investigated by placing BR in a lipid matrix where it will execute fast rotational diffusion. From the rotationally averaged powder patterns the orientations of sidechains with respect to the diffusion axis can be determined. The anion binding site of HR will be studied as will the possibility of isotopically labelling the protein.

  Funding Agency

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
5R01GM023289-13
Application #
3271575
Study Section
Biophysics and Biophysical Chemistry B Study Section (BBCB)
Project Start
1976-05-01
Project End
1990-06-30
Budget Start
1988-07-01
Budget End
1989-06-30
Support Year
13
Fiscal Year
1988
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

Showing the most recent 10 out of 38 publications