Orientation Distributions for Cytochrome c on Polar and Nonpolar Interfaces by Total Internal Reflection Fluorescence

Abstract

The formation of chemisorbed monolayers of yeast cytochrome c on both uncharged polar and nonpolar soft surfaces of organic self-assembled monolayers (SAM) on solid inorganic substrates was followed in situ by polarized total internal reflection fluorescence. Two types of nonpolar surfaces and one type of uncharged polar surface were used. The first type of nonpolar surface contained only thiol endgroups, while the other was composed of a mixture of thiol and methyl endgroups. The uncharged polar surface was provided by the mixture of thiol and hydroxyl endgroups. The thiol endgroups were used to form a covalent disulfide bond with the unique surface-exposed cysteine residue 102 of the protein. The mean tilt angle of the protein's zinc-substituted porphyrin was found to be 41 degrees and 50 degrees for the adsorption onto the nonpolar and uncharged polar surfaces, respectively. The distribution widths for the pure thiol and the thiol/methyl and thiol/hydroxyl mixtures were 9 degrees, 1 degrees, and 18 degrees, respectively. The high degree of the orientational order and good stability achieved for the protein monolayer on the mixed thiol/methyl endgroup SAM makes this system very attractive for studies of both intramolecular and intermolecular electron transfer processes.

Fluorescence Spectroscopy in Biology

“…For example, Balakrishnan [136] showed the influence of spatial curvature effects on FCS of molecules diffusing within membranes, and Chirico et al [137] modelled a similar although small effect for dyes in solution that was earlier observed by Osborne et al [138]. The final result of an FCS experiment is the autocorrelation function that is condensed out of a large file of recorded fluorescence intensities.…”

“…Recent advances in ultra-sensitive instrumentation have allowed the detection of individual atoms and molecules in solids [133,134], on surfaces [135,136], and in the condensed phase [137,138] using laser-induced fluorescence. In particular, single molecule detection in the condensed phase enables scientists to explore new frontiers in many scientific disciplines, such as chemistry, molecular biology, molecular medicine and nanostructure materials.…”

“…In the simplest form of this method, the linear absorption dichroism is measured by examining the dependence of the evanescently excited fluorescence on the polarization of the evanescent field. P-TIRFM has been experimentally applied to a variety of fluorescent lipids in supported planar membranes [91,129,[131][132][133] as well as cytochrome c at dif-ferent surfaces [134][135][136][137][138] and plasminogen at modified surfaces [139]. 6.1).…”

Scanning Electrochemical Microscopy of Cytochrome c Peroxidase through the Orientation‐Controlled Immobilisation of Cytochrome c

“…The most common covalent immobilisation approach is to form an amide linkage between a carboxyl‐terminated (−COOH) self‐assembled monolayer (SAM) and lysine residues (LYS‐73, LYS‐86 or LYS‐87) of the cyt c . However, a major issue with amide linkages is the lack of chemoselectivity for specific lysine residues . Figure a highlights all lysines present on iso‐1 cyt c , where poor chemoselectivity can result in cyt c conjugation to the surface with a number of orientations .…”

Specific Vectorial Immobilization of Oligonucleotide‐Modified Yeast Cytochrome c on Carbon Nanotubes

“…[29] The properties of vectorially orientated YCC, chemisorbed via Cys102 at thiol-modified surfaces, have been extensively studied. [30][31][32][33] Scanning probe microscopy has shown that YCC can be immobilized directly on gold via Cys102. [34][35][36] Moreover, we have shown that YCC, vectorially chemisorbed on gold, exhibits very fast, reversible interfacial electron transfer and retains its native functionality, that is, the ability to interact with redox enzymes in solution.…”