7.2 Site-directed mutagenesis
The application of site-directed mutagenesis (SDM) to the study of protein function has been illustrated with the enzyme lysozyme, as described previously. SDM is a very powerful technique in the study of protein function, allowing the experimenter to assess the importance of particular amino acid side-chains in a protein. It is most commonly used in the study of enzymes; however, it is also very useful in identifying key residues in protein–protein interactions. In this section, we will consider the methodology and design of SDM studies.
Provided that the gene encoding the protein of interest is available and there is a suitable system for expressing the gene, it is possible, using recombinant DNA technology, to produce a mutant protein in which a specific amino acid has been replaced with a different amino acid. To produce the desired mutant protein, it is necessary to change a single base in the gene encoding the protein of interest, such that the codon for the target amino acid is changed to that encoding the desired replacement amino acid.
One of the principal methods that is used for SDM is based on a technique called primer extension. A typical site-directed mutagenesis procedure, using this method, is outlined in Figure 48. The coding sequence of the normal gene is first cloned into a plasmid vector that can exist as single-stranded DNA (ssDNA), but which, during replication in a host cell, goes through a double-stranded form (dsDNA). A short DNA oligonucleotide (10–20 nucleotides in length) is synthesised that is complementary to the region of the gene to be mutated, except for the single base change in the appropriate codon (X in Figure 48); for example, changing TTG, the codon for Gln, to TTC, the codon for Glu. The oligonucleotide is annealed to the ssDNA form of the cloned sequence and serves as a primer for DNA synthesis by DNA polymerase. After synthesis, the two ends of the new strand are joined using a DNA ligase enzyme and this double-stranded DNA (termed a heteroduplex) is used to transform host bacteria. The vector (with its insert) is replicated in the host and mutant clones can be selected on the basis of their ability to hybridise to the original oligonucleotide primer under suitable discriminating conditions. The mutant gene can be subcloned into an appropriate vector and expressed in large quantities in transformed cells. The resulting mutant protein can then be analysed with regard to its structure and function (e.g. catalytic activity) and comparisons made with the normal (wild-type) protein.
Given the number of amino acids in an average protein and the possibility of substituting each residue with a choice of 19 other amino acids, it is clear that SDM experiments require careful design. There are two main questions that should be considered in designing a strategy for SDM. Firstly, which amino acid should be mutated, and secondly, which amino acid should it be replaced with? It is also possible to produce mutant proteins in which two or even three residues have been mutated.
The selection of an amino acid for mutation is often based on detailed knowledge of the protein's three-dimensional structure. For example, from the X-ray structure of an enzyme with a bound substrate analogue, amino acids likely to be involved in binding the substrate or catalysing the reaction might be identified on the basis of their proximity to the substrate and their chemical properties. These residues are interesting targets for mutagenesis. Similarly, identification of highly conserved residues in comparisons of amino acid sequences of related proteins can indicate important roles for these in the protein and can form the basis for an informed choice of target residues for mutagenesis.
Having identified the amino acid that we want to mutate, the choice of replacement must then be considered. In Table 6, some amino acid replacements commonly used in SDM are listed. The choice of replacement generally depends on the supposed role of the residue in question. For example, to test the importance of a particular residue in an enzyme mechanism, it could be replaced with one of similar overall size but differing in its chemical characteristics.
Table 6 Some of the common amino acid replacements used in SDM experiments.
|Ala||Ser, Gly, Thr|
|Arg||Lys, His, Gln|
|Asn||Asp, Gln, Glu, Ser, His, Lys|
|Asp||Asn, Gln, Glu, His|
|His||Asn, Asp, Gln, Glu, Arg|
|Leu||Met, Ile, Val, Phe|
|Lys||Arg, Gln, Asn|
|Ser||Ala, Thr, Asn, Gly|
|Tyr||Phe, His, Trp|
Why do you think it would be advisable to use a residue of similar size for such a substitution?
Matching the residue for size would minimise any steric effects that might alter the overall structure and thereby confound observation of the effect of changes to the chemistry of the residue.
For example, Glu could be replaced by Gln, which is similar in size and shape but lacks the negative charge. (This was one of the mutations used to determine the role of Glu 35 in the catalytic mechanism of lysozyme; see Section 6.3.) Substitution of a particular amino acid residue with a larger residue can result in steric interference and should be avoided. It is preferable to use residues that are smaller than the wildtype residue, as they tend not to disrupt the overall structure of the protein. Proline is commonly substituted for amino acids in parts of the protein that are thought to have key structural roles or to undergo some structural rearrangements as a part of normal functioning. The introduction of proline in such a position affects the geometry of the polypeptide backbone, usually causing a bend, and rendering it relatively inflexible.