Sample electron-transfer proteins

Bill Heineman s group developed an elegant indirect titration method in the 1970s [15], Indirect coulometric titrations and optically transparent thin-layer electrochemical cells were combined to provide a simple and quick means of making formal potential measurements on electron transfer proteins. Moreover, the amount of sample required for this measurement was quite small. This is now a routine method for measuring the formal potential of electron transfer proteins, which is used by a wide range of non-electrochemical scientists. Use of this method in our laboratory, greatly facilitated by help from the Heineman laboratory, led to our later efforts to develop direct electfochemical methods for protein and enzyme studies. 

Spin, Mossbauer, fluorescent and phosphorescent labels were introduced into the various portions of the system being studied. They were covalently bound to the RC surface groups, adsorbed by the hydrophobic segments of the protein and membrane, and 57Fe atoms were incorporated by way of biosynthesis into iron-containing proteins. Then, in the same samples, the dependence on temperature, moisture content and viscosity was measured for the label mobility and the rate constant of electron transfer... 

The introduction and implementation of heteronuclear-based multidimensional techniques have revolutionized the protein NMR field. Large proteins (> 100 residues) are now amenable to detailed NMR studies and structure determination. These techniques, however, necessarily require a scheme by which and isotopes can be incorporated into the protein to yield a uniformly labeled sample. Additional complications, such as extensive covalent post-translational modifications, can seriously limit the ability to efficiently and cost effectively express a protein in isotope enriched media - the c-type cytochromes are an example of such a limitation. In the absence of an effective labeling protocol, one must therefore rely on more traditional proton homonuclear NMR methods. These include two-dimensional (1) and, more recently, three-dimensional H experiments (2,3). Cytochrome c has become a paradigm for protein folding and electron transfer studies because of its stability, solubility and ease of preparation. As a result, several high-resolution X-ray crystal structure models for c-type cytochromes, in both redox states, have emerged. Although only subtle structural differences between redox states have been observed in these... 

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