Early and late M intermediates in the bacteriorhodopsin photocycle: a solid-state NMR study.
To enforce vectorial proton transport in bacteriorhodopsin (bR), it is necessary that there be a change in molecular structure between deprotonation and reprotonation of the chromophore-i.e., there must be at least two different M intermediates in the functional photocycle. We present here the first detection of multiple M intermediates in native wild-type bacteriorhodopsin by solid-state NMR. Illumination of light-adapted [zeta-15N-Lys]-bR at low temperatures shifts the 15N signal of the retinal Schiff base (SB) downfield by about 150 ppm, indicating a deprotonated chromophore. In 0.3 M Gdn-HCl at pH 10.0, two different M states are obtained, depending on the temperature during illumination. The M state routinely prepared at the lower temperature, Mo, decays to the newly observed M state, Mn, and the N intermediate, as the temperature is increased. Both relax to bR568 at 0 degreesC. A unique reaction sequence is derived: bR568-->Mo-->(Mn+N)-->bR568. Mo and Mn have similar chemical shifts at [12-13C]ret, [14-13C]ret, and [epsilon-13C]Lys216, indicating that Mn, like Mo, has a 13-cis and C=N anti chromophore. However, a small splitting in the [14-13C]ret signal of Mo reveals that it has at least two substates. The 7 ppm greater shielding of the SB nitrogen in Mn compared to Mo suggests an increase in basicity and/or hydrogen bonding. Probing the peptide backbone of the protein, via [1-13C]Val labeling, reveals a substantial structural change between Mo and Mn including the relaxation of perturbations at some sites and the development of new perturbations at other sites. The combination of the change in the protein structure and the increase in the pKa of the SB suggests that the demonstrated Mo-->Mn transition may function as the "reprotonation switch" required for vectorial proton transport.
Hu
J G
JG
Department of Chemistry, Keck Institute for Cellular Visualization, Brandeis University, Waltham, Massachusetts 02254-9110, USA.
Sun
B Q
BQ
Bizounok
M
M
Hatcher
M E
ME
Lansing
J C
JC
Raap
J
J
Verdegem
P J
PJ
Lugtenburg
J
J
Griffin
R G
RG
Herzfeld
J
J
D001436:Bacteriorhodopsins / Q000737:chemistry*; D002247:Carbon Isotopes; D019791:Guanidine; D019617:Halobacterium salinarum; D008027:Light; D008239:Lysine / Q000737:chemistry; D009682:Magnetic Resonance Spectroscopy; D009587:Nitrogen Isotopes; D010777:Photochemistry; D011392:Proline / Q000737:chemistry; D011487:Protein Conformation; D011522:Protons; D012545:Schiff Bases; D013053:Spectrophotometry
D002247:Carbon Isotopes; D009587:Nitrogen Isotopes; D011522:Protons; D012545:Schiff Bases; D001436:Bacteriorhodopsins; D011392:Proline; D019791:Guanidine; D008239:Lysine