The objective of this work is to covalently attach bacteriorhodopsin (BR) to a gold surface via genetic substitution of cysteine for serine (S35C) at the 35th amino acid position. Samples of BR-containing purple membrane (PM) on gold were evaluated using atomic force microscopy (AFM) and x-ray photoelectron spectroscopy (XPS). AFM images reveal a surface coverage of S35C-containing PM fragments of approximately 25%. XPS measurements reveal a small excess of sulfur for S35C-containing PM on gold, and a much larger excess of sulfur on wildtype-containing PM on gold. In both cases, the excess sulfur is covalently bound to the gold surface and appears to originate from dissociated methyl mercaptan groups from methionine residues on the external surfaces of BR. We conclude that the quantity of excess sulfur is smaller for S35C than for wildtype because the S35C’s sulfhydryl binds some PM fragments to the surface, reducing the quantity of methyl mercaptan that can bind to the surface. It then follows that the rather low coverage of S35C-containing PM fragments on gold is due to interference of the methyl mercaptan groups with the binding of S35C-containing PM fragments to the surface. Coverage might be increased by immobilizing S35C-containing fragments on a functionalized surface using a heterobifunctional cross-linker, thereby preventing dissociation of methyl mercaptan groups from BR, and by using smaller PM fragments.
Purple membrane is a constituent of the cell membrane of Halobacterium halobium. It is of technological interest due to the very fast, efficient electrical response to light of its integral protein, bacteriorhodopsin. Dried films of purple membrane are of importance for the fabrication of devices from this material. In this article, x-ray photoelectron spectroscopy (XPS) spectra of purple membrane fragments dried on SiO2 are presented. To remove excess lipid from the substrate surface, the samples were washed with distilled water subsequent to the purple membrane drying step. The presence of purple membrane fragments on the substrate surface was verified by atomic force microscopy prior to XPS analysis.
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