Protein structure analysis selection

The advent of molecular engineering has prompted new developments in the area of protein structural analysis with the preparation of protein mutants by site-directed mutagenesis. In this manner, a reactive amino acid residue, generally a cysteine, may be introduced at a well chosen location, providing a heavy atom binding site. There is thus a need to provide heavy atom reagents that will specifically target selected protein residues. 

In this chapter selected examples from our group are discussed to show how metal coordination to ligand-modified amino acids or peptides can be used for induction or fixing of defined conformations in amino acid residues or di- and tripeptides. In this context Ramachandrarfs method for conformational analysis of peptide or protein structures will be introduced. 

Directed evolution as a tool to probe the basis of protein structure, stability, and function is in its infancy, and many fruitful avenues of research remain to be explored. Studies so far have focused on proteins that unfold irreversibly, making detailed thermodynamic analysis impossible. The application of these methods to reversibly folding proteins could provide a wealth of information on the thermodynamic basis of high temperature stability. A small number of studies on natural thermophilic proteins have identified various thermodynamic strategies for stabilization. Laboratory evolution makes it possible to ask, for example, whether proteins have adopted these different strategies by chance, or whether certain protein architectures favor specific thermodynamic mechanisms. It will also be possible to determine how other selective pressures, such as the requirement for efficient low temperature activity, influence stabilization mechanisms. The combination of directed evolu-... 

Directed evolution and antibody affinity maturation offer efficient routes to redesigning proteins for new functional characteristics. Adaptive mutations and well-defined selection pressures allow structural analysis of the evolved products to provide insights into the molecular basis of protein structure and function. It is interesting to note that the majority of mutations that were obtained in the present maturation and directed evolution experiments were located at positions away from the enzymatic active sites. Perhaps this is due to the inherent difficulty in retaining catalytic activity with most active site amino acid substitutions. 

Because a library can contain thousands of different clones, it can be difficult to isolate a clone with DNA of specific interest. This is because the majority of cloned DNAs do not contain a readily selectable genetic marker, such as antibiotic resistance, or lead (as discussed earlier) to the production of a foreign protein. Methods to achieve this have been developed and utilize hybridization, immunochemical, and structural techniques. A specific DNA sequence of only several kilobases can be isolated from a genome containing in excess of 106 kilobases. Hybridization requires a radioactive DNA or RNA molecule (a probe) that is complementary (or partially so) to the sequence of the cloned DNA. Immunologic techniques require that the polypeptides coded by the DNAs of interest are available and have specific antigenic properties that allow detection. Structural analysis can also be used when the other techniques are inapplicable. 

The Streptomyces lividans K+ channel (KcsA) is a 160-residue protein that forms homotetrameric channels closely related to the pore domain of larger voltage-dependent channels (Schrempf et al., 1995). When purified and reconstituted in lipid bilayers, KcsA catalyzes single-channel activities with selectivity properties identical to those of other eukaryotic K+ channels (Cuello et al., 1998 Heginbotham et al., 1999 Meuser et al., 1999). The fact that KcsA is easily expressed in Escherichia coli at milligram levels made this protein an ideal target for structural analysis. 

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