Site directed spin labeling studies of structure and dynamics in bacteriorhodopsin.
Site-directed spin labeling of membrane proteins has been used to determine: (1) the topography of the polypeptide chain with respect to the membrane/solution interface, and (2) the identity and orientation of secondary structure in selected regions. These features are deduced from the collision rates of nitroxide side chains with paramagnetic reagents in solution, and the principles of the method are reviewed with reference to bacteriorhodopsin. The dynamics of the nitroxide side chains relative to the backbone reveal tertiary interactions of the labeled site, and provide a promising means of time-resolving conformational changes. This aspect is illustrated by recent studies of structural changes in bacteriorhodopsin during the photocycle. In these experiments, nitroxide side chains were introduced at residues 72, 101 and 105 after replacement of the original residues by cysteine. Upon flash photolysis, the electron paramagnetic resonance spectrum of a nitroxide at 101, but not those at 72 or 105, is time-dependent. The spectral change develops during the decay of the M-intermediate, and reverses upon return to the ground state. The results suggest a movement of the C-D or E-F interhelical loops during the protonation changes of aspartate 96.
Steinhoff
H J
HJ
Institut für Biophysik, Ruhr-Universität Bochum, Germany.
Mollaaghababa
R
R
Altenbach
C
C
Khorana
H G
HG
Hubbell
W L
WL
D001436:Bacteriorhodopsins / Q000737:chemistry* / Q000378:metabolism*; D001665:Binding Sites; D004578:Electron Spin Resonance Spectroscopy / Q000379:methods; D007700:Kinetics; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed; D017354:Point Mutation; D011487:Protein Conformation; D017433:Protein Structure, Secondary; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D012996:Solutions; D013113:Spin Labels; D013997:Time Factors
D011994:Recombinant Proteins; D012996:Solutions; D013113:Spin Labels; D001436:Bacteriorhodopsins