Time-resolved site-directed spin-labeling studies of bacteriorhodopsin: loop-specific conformational changes in M.
A spin-label at site 101 in the C-D loop of bacteriorhodopsin was previously found to detect a conformational change during the M --> N transition [Steinhoff, H. -J., Mollaaghababa, R., Altenbach, C., Hideg, K., Krebs, M. P., Khorana, H. G., and Hubbell, W. L. (1994) Science 266, 105-107]. We have extended these time-resolved electron paramagnetic resonance studies in purple membranes by analyzing conformational changes detected by a spin-label at another site in the C-D loop (103), and at sites in the A-B loop (35), the D-E loop (130), and the E-F loop (160). In addition, we have investigated the motion detected by a spin-label at site 101 in a D96A mutant background that has a prolonged M intermediate. We find that among the examined sites, only spin-labels in the C-D loop detect a significant change in the local environment after the rise of M. Although the D96A mutation dramatically prolongs the lifetime of the M intermediate, it does not perturb either the structure of bacteriorhodopsin or the nature of the light-activated conformational change detected by a spin-label at site 101. In this mutant, a conformational change is detected during the lifetime of M, when no change in the 410 nm absorbance is observed. These results provide direct structural evidence for the heterogeneity of the M population in real time, and demonstrate that the motion detected at site 101 occurs in M, prior to Schiff base reprotonation.
Mollaaghababa
R
R
Departments of Biology and Chemistry, Massachusetts Institute of Technology, Cambridge, Massachusetts 02139, USA.
Steinhoff
H J
HJ
Hubbell
W L
WL
Khorana
H G
HG
D000409:Alanine / Q000235:genetics; D000595:Amino Acid Sequence; D019943:Amino Acid Substitution / Q000235:genetics; D001224:Aspartic Acid / Q000235:genetics; D001436:Bacteriorhodopsins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D003545:Cysteine / Q000235:genetics; D004578:Electron Spin Resonance Spectroscopy / Q000379:methods; D004591:Electrophoresis, Polyacrylamide Gel; D007700:Kinetics; D008698:Mesylates / Q000737:chemistry; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D010782:Photolysis; D011487:Protein Conformation; D017433:Protein Structure, Secondary; D018717:Purple Membrane / Q000737:chemistry / Q000378:metabolism; D013056:Spectrophotometry, Ultraviolet; D013113:Spin Labels
D008698:Mesylates; D013113:Spin Labels; D001224:Aspartic Acid; C074502:methanethiosulfonate; D001436:Bacteriorhodopsins; D003545:Cysteine; D000409:Alanine