Virtual mutagenesis

How is the binding specificity of the heterodimer achieved compared with the specificity of Mat a2 alone The crystal structure rules out the simple model that the contacts made between the Mat a2 homeodomain and DNA are altered as a result of heterodimerization. The contacts between the Mat o2 homeodomain and DNA in the heterodimer complex are virtually indistinguishable from those seen in the structure of the Mat o2 monomer bound to DNA. However, there are at least two significant factors that may account for the increased specificity of the heterodimer. First, the Mat al homeodomain makes significant contacts with the DNA, and the heterodimeric complex will therefore bind more tightly to sites that provide the contacts required by both partners. Second, site-directed mutagenesis experiments have shown that the protein-protein interactions involving the... 

There have been two independent mutagenesis studies that have been directed toward probing the role of E4 in the PLCB(. reaction [36, 94]. In the first of these, the kinetic parameters kcM and Km of the E4L, E4D, and E4Q mutants, which each gave CD spectra similar to wild-type, were determined by the choline quantitation method [33], and these mutants were found to retain 6-60% of the catalytic efficiency (i.e., kcat/iCm) of wild-type [36, 64]. Furthermore, the pH-dependence with activity curve of the E4L mutant was virtually identical with that for E146Q (Fig. 13) and similar to that of wild-type. In the other inves-... 

Unstable proteins may be modified by the molecular biological technique of site-directed mutagenesis to remove the site of instability— for instance, an oxidizable cysteine. Such techniques are appropriate for commercial production of proteins, but may of course alter natural functioning parameters. Increased thermostability can be one modification, although it is not easy to predict mutations that will improve that parameter. Thermostable proteins originating from thermophilic bacteria do not need structural modification and, if expressed in large amounts, can be purified satisfactorily in one step by simply heat-treating the extract at 70°C for 30 min, which denatures virtually all the host proteins (e.g., see Oka et al., 1989). 

A commonly used test in experimental animals is the dominant-lethal test, generally assumed to monitor the induction of chromosomally abnormal sperm. There are reports of loss of fertility of human males exposed to various chemicals. It is difficult to attribute such effects to genetic events, rather them to cellular effects. An increase in genetically defective sperm, detected by a decrease in fertility, would require a very large and carefully controlled study. The use of birth control and the changing social patterns of family size virtually rule out evidence on fertility as useful in monitoring mutagenesis. 

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. 

Related methods for the prediction of hotspots on protein-protein interfaces using virtual mutagenesis and virtual alanine scanning have recently been reviewed by DeLano [34],... 

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