Sulfur distribution in bacteriorhodopsin from multiple wavelength anomalous diffraction near the sulfur K-edge with synchrotron x-ray radiation

Bacteriorhodopsin contains nine sulfur atoms from the nine methionine residues. The distribution of these sulfur atoms in the projected density map was determined from x-ray diffraction experiments using multiple wavelength anomalous diffraction (MAD) at the sulfur K-edge (5.02 Angstrom) with synchrotron radiation. The experiments were performed with uniaxial samples of oriented purple membranes at room temperature and 86% relative humidity. For such samples only the real part f' (lambda) of the resonant scattering amplitude of sulfur contributes to the observed scattering intensity. The sulfur density was determined from the difference in diffraction intensities detected at two wavelengths near the sulfur K-edge that were similar to 0.004 Angstrom apart. The measured change in f' between these two wavelengths corresponds to 6 electron units. This shows that large anomalous dispersion effects occur near the sulfur K-edge, The in-plane positions of the sulfur atoms of Met(32), Met(56), and Met(209) were determined unambiguously. The difference density from Met(20), Met(60), Met(118), and Met(145) is concentrated in the interior of the seven alpha-helical bundle, overlaps strongly in the projected density map, and cannot be resolved at the resolution of these experiments (8.2 Angstrom). This method of localizing individual sulfur atoms can be applied to other two-dimensional protein crystals and is promising in conjunction with the site-directed introduction of sulfur atoms by the use of cysteine mutants.