The bacteriorhodopsin structure, deduced from the electron microscopy data of two-din~ensional crystals in the purple membrane and most recently from X-ray data of microcrystals grown in lipidic cubic phases, shows that in the resting state of the protein, most of the ionizable residues (Asp, Glu, Arg and Lys) are very close to or at the two membrane surfaces, with the exception of Asp-l 15, Asp-2 12, Asp-85 and Arg-82. The last three residues, together with one or more water molecules, form the complex counterion of the protonated Schiff base (SB). CP/MAS studies of the proton-motive photo-cycle of [N1 2-5N2]Arg-bR in 0.1 M NaCl, pH=lO have identified an arginine residue whose environment changes in the transition between the bR568 and the early M states. This change persists in the late M state, and manifests itself by the appearance of two 'wing peaks, separated by 24 ppm, that flank the central resonance of the other six arginine sidechains. The same signal for an arginine iii a highly asymmetric environment is reproduced in the yellow M-like (0.3 M Gdn.HCJ, pH=l0.8) and the blue 0-like (0.1 M NaCl, pH=6.5) dark-adapted forms of the D85N mutant. The similarities between the wild type M state and the D85N alkaline form, which originate from the deprotonation of the SB and the neutrality of residue 85, suggest that the 'wing peaks arise from the nearby R82. Additional evidence comes from the delayed CP experiment, which isolates signals from nuclei cross polarized from water exchangeable protons. The 'wingt peaks are missing from the delayed CP spectrum of the M-like D85N, which indicates that they do not belong to a surface residue. This assignment is consistent with participation of R82 in the Schiff base counterion complex in bR568 (together with D85 and D2 12), and reorientation of R82 towards the extracellular (EC) side of the protein in the M state. This interpretation is also consistent with the effects of R82 mutations on the photo-cycle kinetics and with the notion that R82 controls the pKa s of D85 and the proton release group at the EC. Since the protonation state of the SB does not seem to affect the 'wing peaks in D85N (the SB is protonated at pH=6.5 and deprotonated at pH=lO.8), studies to clarify the chemical environment of the guanidyl nitrogens in the N state of the wild type photo-cycle are under way.

Agency
National Institute of Health (NIH)
Institute
National Center for Research Resources (NCRR)
Type
Biotechnology Resource Grants (P41)
Project #
5P41RR000995-23
Application #
6279708
Study Section
Project Start
1998-05-01
Project End
1999-04-30
Budget Start
1997-10-01
Budget End
1998-09-30
Support Year
23
Fiscal Year
1998
Total Cost
Indirect Cost
Name
Massachusetts Institute of Technology
Department
Type
DUNS #
City
Cambridge
State
MA
Country
United States
Zip Code
02139
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