Site-specific mutagenesis principle

The following is a brief summary of the techniques and problems particularly associated with nucleotide sequencing and molecular cloning. Readers are advised to consult other references for more exhaustive treatment of these and other subjects regarding principles and methods (3,4,67) and step-by-step procedures (15). For principles and methods of PCR-based site-specific mutagenesis, refer to Appendix A. 

The principle advantage of the physical labeling method is the possibility of receiving direct information about the structure, mobility and local micropolarity of certain parts of a molecular object of any molecular mass. Developments in synthetic chemistry, biochemistry and site-directed mutagenesis have provided researchers with a wide assortment of labels and probes, and have paved the way for the specific modification of protein function groups, including enzyme active sites. 

The time is ripe for the widespread application of biocatalysis in industrial organic synthesis and according to a recent estimate [113] more than 130 processes have been commercialised. Advances in recombinant DNA techniques have made it, in principle, possible to produce virtually any enzyme for a commercially acceptable price. Advances in protein engineering have made it possible, using techniques such as site directed mutagenesis and in vitro evolution, to manipulate enzymes such that they exhibit the desired substrate specificity, activity, stability, pH profile, etc. [114]. Furthermore, the development of effective immobilisation techniques has paved the way for optimising the performance and recovery and recycling of enzymes. 

The problem with the application of fliis principle in proteins is spectral crowding for proton interactions and, combined with this, only partial definition of hyperfine lines over the whole range of flie EPR spectrum. This makes direct tensor determination somewhat ambiguous, but in connection with known molecular stractures a unique solution can typically be found. A very important asset in this respect is site-directed mutagenesis, by which specific amino acid residues can be changed. For most of the examples of iron-sulfur clusters discussed, a full analysis was possible even without this tool. 

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