Proteins stability site-directed mutagenesis

Since there are strict stereochemical requirements for the relative positions and orientations of the two participating cysteine residues,11 addition of new disulfides to existing proteins by site-directed mutagenesis has not always produced the desired increase in stability. Introduction of disulfide bonds has been attempted for phage T4 lysozyme,4-71 phage A repressor,81 dihydrofolate reductase,91 and subtilisins.10-131 Among them the most extensive study has been performed on T4 lysozyme, and enhancement of protein stability has been successful. 

In summary, we have shown that site-directed mutagenesis is an important tool which allows precise investigation into the detailed chemistry of protein-protein interaction. HEL has proved to be ideal for these studies. Monoclonal antibodies, X-ray crystal structures of protein complexes, and evolutionary lysozyme variants found in nature provide an excellent system for understanding the molecular basis of protein recognition. Site-directed mutagenesis offers the ability to alter a protein at a specific site. Interpretation of the results within the context of an X-ray crystal structure allows the quantitative assignment of the free energy contributions from individual amino adds to the stability of the complex. 

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. 

The family of serine proteases has been subjected to intensive studies of site-directed mutagenesis. These experiments provide unique information about the contributions of individual amino acids to kcat and KM. Some of the clearest conclusions have emerged from studies in subtilisin (Ref. 9), where the oxyanion intermediate is stabilized by t>e main-chain hydrogen bond of Ser 221 and an hydrogen bond from Asn 155 (Ref. 2). Replacement of Asn 155 (e.g., the Asn 155— Ala 155 described in Fig. 7.9) allows for a quantitative assessment of the effect of the protein dipoles on Ag. ... 

A variety of approaches exist for stabilizing proteins, for example, chemical modification, immobilization, and site-directed mutagenesis [95,96], but these techniques are not within the scope of this chapter. The focus here will be on stabilization of proteins via formulation development. The principal formulation strategy is to stabilize the protein using clinically acceptable additives (excipients) or through the use of suitable pharmaceutical-processing technologies. 

High temperatures can break native S-S bonds and form new S-S bonds which can lock the protein into a denatured eonfiguration [89]. Low pH, sodium dodecyl sulfate. Tween 80, chaotropie salts, and exogenous proteins have been used to protect proteins from thermal inaetivation [90]. Ethylene glycol at 30-50% was used to protect the antiviral activity of P-interferon preparations [91]. Human serum albumin was used in recombinant human interferon-Psei-n which resulted in increased thermal stability [62]. Water-soluble polysaeeharides sueh as dextrans and amylose [92], as well as point-specific (site-directed) mutagenesis [93] have also been used to increase thermal stability of therapeutie proteins and peptides. 

Further advantages of biocatalysis over chemical catalysis include shorter synthesis routes and milder reaction conditions. Enzymatic reactions are not confined to in vivo systems - many enzymes are also available as isolated compounds which catalyze reactions in water and even in organic solvents [28]. Despite these advantages, the activity and stability of most wild-type enzymes do not meet the demands of industrial processes. Fortunately, modern protein engineering methods can be used to change enzyme properties and optimize desired characteristics. In Chapter 5 we will outline these optimization methods, including site-directed mutagenesis and directed evolution. 

In the case of oligomeric proteins in which subunit contact regions have been revealed by X-ray crystallography34 353 or other methods described above,363 the equilibrium between oligomer and monomer can be changed by site-directed mutagenesis. For example, stable monomers of tyrosyl-tRNA synthetase were produced by a mutation of Phe-164 at the subunit interface to Asp, and it was revealed that the monomers are inactive and do not bind the substrate tyrosine.343 In the case of yeast triosephosphate isomerase, replacement of Asn-78 at the subunit interface did not cause dissociation of subunits under normal conditions.353 However, the stability of the enzyme was significantly lowered by the mutation, probably due to decreased subunit-subunit interaction.353... 

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