Site-directed spin-labeling reveals the orientation
of the amino acid
side-chains in the E-F loop of bacteriorhodopsin.
Pfeiffer M, Rink T, Gerwert K, Oesterhelt D, Steinhoff HJ
Lehrstuhl fur Biophysik, Ruhr-Universitat Bochum, Bochum, 44780, Germany.
Due to high temperature factors and the lack of considerable electron
density, electron microscopy and X-ray
experiments on the cytoplasmic E-F loop of bacteriorhodopsin result in a variety of structural models. As the
experimental conditions regarding ionic strength, temperature and the presence of detergents may affect the structure
of the E-F loop, we employ electron paramagnetic resonance and site-directed spin-labeling to study the structure of
this loop under physiological conditions. The amino acid residues at positions 154 to 171 were replaced by cysteine
residues and derivatized with a sulfhydryl-specific nitroxide spin label one by one. The conventional and power
saturation electron paramagnetic spectroscopy provide the mobility of the nitroxide and its accessibility to dissolved
molecular oxygen and membrane-impermeable chromium oxalate in the respective site. The results show that K159
and A168 are located at the water-lipid interface of helices E and F, respectively. The orientation of the amino acid
side-chains in the helical regions from positions 154 to 159 and 166 to 171 were found to agree with published
structural data for bacteriorhodopsin. In the residue sequence from positions 160 to 165 the EPR data yield evidence
for a turned loop structure with the side-chains of M163 and S162 oriented towards the proton channel and the water
phase, respectively. Copyright 1999 Academic Press.