Protein Engineering mutant proteins

Subtilisins are a group of serine proteinases that are produced by different species of bacilli. These enzymes are of considerable commercial interest because they are added to the detergents in washing powder to facilitate removal of proteinaceous stains. Numerous attempts have therefore recently been made to change by protein engineering such properties of the subtilisin molecule as its thermal stability, pH optimum, and specificity. In fact, in 1988 subtilisin mutants were the subject of the first US patent granted for an engineered protein. 

The subtilisin mutants described here illustrate the power of protein engineering as a tool to allow us to identify the specific roles of side chains in the catalytic mechanisms of enzymes. In Chapter 17 we shall discuss the utility of protein engineering in other contexts, such as design of novel proteins and the elucidation of the energetics of ligand binding to proteins. 

Protein engineering is now routinely used to modify protein molecules either via site-directed mutagenesis or by combinatorial methods. Factors that are Important for the stability of proteins have been studied, such as stabilization of a helices and reducing the number of conformations in the unfolded state. Combinatorial methods produce a large number of random mutants from which those with the desired properties are selected in vitro using phage display. Specific enzyme inhibitors, increased enzymatic activity and agonists of receptor molecules are examples of successful use of this method. 

Many proteins currently selected for structural study crystallize only with small sample volume. This is especially true in the case of novel engineered mutants or... 

The fastest-folding small proteins generally fold on much slower time scales than the time scale of formation of secondary structure. The speed record is currently held by lambda(6-85), a truncated, monomeric form of the N-terminal domain of lambda repressor, which refolds with a half-life of approximately 140 fjis. A thermostable lambda(6-85) variant with alanine substituted for glycine residues 46 and 48 in the third helix folds faster in dilute solutions of de-naturant, with an extrapolated half-life of less than 10 /us in water.13 Cold-shock protein CspB from Bacillus subtilis folds in about 1 ms.61 Engineered mutants of the P22 Arc repressor62 and CI263 fold in a fraction of a millisecond. 

The contribution of electrostatic interactions to fast association was analyzed by applying the classical Debye-Hiickel theory of electrostatic interactions between ions to mutants of bamase and barstar whose ionic side chains had been altered by protein engineering (Chapter 14).16 The association fits a two-step model that is probably general (equation 4.84). 

The production of mutants by site-specific mutagenesis is the basis of the rational and systematic analysis of proteins and their redesign by protein engineering, as described in the next chapter for the tyrosyl-tRNA synthetase and subtilisin. 

The more recent structural evidence supports the role of substrate distortion. A trisaccharide in the BCD sites of crystalline hen egg white lysozyme is distorted to the half-chair,229 as is a substrate bound to the Asp-52 — Ser mutant studied in solution by NMR.230 Protein engineering studies on the residues involved support the conclusions.231 A chitobiose bound to a crystalline chitobiase has the equivalent sugar distorted to a sofa.232... 

Chemically Modified Mutants, a Marriage of Chemical Modification and Protein Engineering... 

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