Mutagenesis site directed

One powerful approach towards analysing protein structure and function is by modifying or mutating the amino-acid sequence in a directed manner and then comparing characteristics of the mutant with the wild-type proteins. Before recombinant DNA technology, a number of chemical means were used to modify specific amino acids, or crude methods such as UV radiation were used to introduce random mutations to the DNA coding sequence. However, 

The specificity of trypsin toward peptide bonds adjacent to lysine and arginine has been altered by replacing a glycine residue in the enzyme active site with an alanine, which carries an additional methyl group. This replacement favors the binding of lysine over the somewhat bulkier arginine. 

In the catalytic mechanism of the serine protease subtilisin, the tetrahedral intermediate is believed to be stabilized by a hydrogen bond to the side chain of Asn 155. Replacement of Asn 155 with Gly left the substrate binding unaffected, but inhibited the catalytic step, confirming the proposed mechanism. 

On the basis of chemical modification studies, Tyr 198 of carboxypeptidase A was proposed to act as a proton donor (i.e., a general acid) in the mechanism of catalysis. However, when Tyr 198 was replaced with Phe by means of site-directed mutagenesis, the modified enzyme retained substantial enzymatic activity, indicating that the tyrosyl hydroxyl may not have a specific role in catalysis. 

Question Does the abolition of activity following mutagenesis unequivocally indicate a catalytic role for the mutated residue  

Not necessarily It is possible that replacement of one amino acid with another may abolish specific interactions critical for local folding or even overall protein stability. It is essential in these types of study to monitor protein conformation after mutagenesis. It is even more helpful if detailed structures, either from x-ray crystallography or NMR (Chap. 4), are available for the wild-type and mutant forms of the enzymes. 

A more efficient approach to this sort of study would be to pick out particular amino acids (perhaps those in the enzyme s catalytic site) and replace them with different amino acids in a systematic way. This would of course require mutations in the DNA sequence at particular sites. This process is known as site-directed or site-specific mutagenesis, and there are several different methods of doing it. Two of the more direct methods are described in the following. 

This duplex with mismatches can be transferred into E. coli just as any other plasmid, and there it will replicate. Half of the progeny plasmids will have the original DNA sequence, but the other half will be the result of copying the strand with the altered sequence, and so these progeny will code for the mutant protein. The mutant progeny can be identified by screening with a hybridization probe, and the bacteria harboring them can be used as a source of the mutant protein. 

Synthetic DNA and biology (Nobel Lecture) , Awgevr. Chem., Int. Ed. Engl. 1994, 33, 1214-1221. 

3 Ferrochelatase a new iron sulfur center-containing enzyme 

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Fig. 6. Polymerase chain reaction (PCR) mediated site-directed mutagenesis. The 5 and 3 ends of the nucleotide strands are indicated. The four arrows surrounding the DNA template represent oligonucleotide primers 1—4. See text for discussion.

Q Zeng, ET Smith, DM Kurtz, RA Scott. Protein determinants of metal site reduction potentials. Site directed mutagenesis studies of Clostridium pasteurianum laibredoxin. Inorg Chim Acta 242 245-251, 1996. 

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... 

Residue 189 is at the bottom of the specificity pocket. In trypsin the Asp residue at this position interacts with the positively charged side chains Lys or Arg of a substrate. This accounts for the preference of trypsin to cleave adjacent to these residues. In chymotrypsin there is a Ser residue at position 189, which does not interfere with the binding of the substrate. Bulky aromatic groups are therefore preferred by chymotrypsin since such side chains fill up the mainly hydrophobic specificity pocket. It has now become clear, however, from site-directed mutagenesis experiments that this simple picture does not tell the whole story. 

Bryan, P., et al. Site-directed mutagenesis and the role of the oxyanion hole in subtilisin. Proc. Natl. Acad. Sci. USA 83 3743-3745, 1986. 

Estell, D.A., Graycar, T.P., Wells, J.A. Engineering an enzyme by site-directed mutagenesis to be resistant to chemical oxidation. /. Biol. Chem. 260 6518-6521, 1985. 

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 specific role of each amino acid residue for the function of the protein can be tested by making specific mutations of the residue in question and examining the properties of the mutant protein. By combining in this way functional studies in solution, site-directed mutagenesis by recombinant DNA techniques, and three-dimensional structure determination, we are now in a position to gain fresh insights into the way protein molecules work. 

Garrett, R. M., and Rajagopalan, K V., 1996. Site-directed mutagenesis of recombinant snlfite oxidase. Journal of Biological Chemistry 271 7387-7391. 

Until recently, the catalytic role of Asp ° in trypsin and the other serine proteases had been surmised on the basis of its proximity to His in structures obtained from X-ray diffraction studies, but it had never been demonstrated with certainty in physical or chemical studies. As can be seen in Figure 16.17, Asp ° is buried at the active site and is normally inaccessible to chemical modifying reagents. In 1987, however, Charles Craik, William Rutter, and their colleagues used site-directed mutagenesis (see Chapter 13) to prepare a mutant trypsin with an asparagine in place of Asp °. This mutant trypsin possessed a hydrolytic activity with ester substrates only 1/10,000 that of native trypsin, demonstrating that Asp ° is indeed essential for catalysis and that its ability to immobilize and orient His is crucial to the function of the catalytic triad. 

M. Smith (University of British Columbia) fundamental contributions to the establishment of oligonucleotide-based, site-directed mutagenesis and its development for protein studies. 

Baldwin, T. O., etal. (1989). Site-directed mutagenesis of bacterial luciferase analysis of the essential thiol. J. Biolumin. Chemilumin. 4 40-48. 

In molecular pharmacology research an indirect proof of a structural model is possible by functional examinations, e.g., by molecular biological experiments. Well-selected site directed mutagenesis and their functional characterization allows confirmation or rejection of a molecular protein model. The process is organized as an iterative procedure, where the biological answer of suggested mutations is used to refine the model. The iteration continues until the model... 

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