Wild-type protein

In Figure 7b, the data are plotted as AG yielding a linear function. Extrapolation to 2ero denaturant provides a quantitative estimate of the intrinsic stability of the protein, AG, which in principle is the free energy of unfolding for the protein in the absence of denaturant. Comparison of the AG values between mutant and wild-type proteins provides a quantitative means of assessing the effects of point mutations on the stability of a protein. 

One of the most important molecular functions of p53 is therefore to act as an activator of p21 transcription. The wild-type protein binds to specific DNA sequences, whereas tumor-derived p53 mutants are defective in sequence-specific DNA binding and consequently cannot activate the transcription of p5 3-con trolled genes. As we will see more than half of the over one thousand different mutations found in p53 involve amino acids which are directly or indirectly associated with DNA binding. 

The results of this careful design of novel disulfide bridges were very encouraging (Figure 17.4). AH the mutants were more stable in their oxidized forms than wild-type protein. The longer the loop between the cysteine... 

Figure 17.14 Model of evolved mutant from cephalosphorinase shuffling. The sequence of the most active cephalosporinase mutant was modeled using the crystal structure of the class C cephalosporinase from Enterobacter cloacae. The mutant and wild-type proteins were 63% identical. This chimeric protein contained portions from three of the starting genes, including Enterobacter (blue), Klebsiella (yellow), and Citrobacter (green), as well as 33 point mutations (red). (Courtesy of A. Crameri.)...

The third reason for favoring a non-radical pathway is based on studies of a mutant version of the CFeSP. This mutant was generated by changing a cysteine residue to an alanine, which converts the 4Fe-4S cluster of the CFeSP into a 3Fe-4S cluster (14). This mutation causes the redox potential of the 3Fe-4S cluster to increase by about 500 mV. The mutant is incapable of coupling the reduction of the cobalt center to the oxidation of CO by CODH. Correspondingly, it is unable to participate in acetate synthesis from CH3-H4 folate, CO, and CoA unless chemical reductants are present. If mechanism 3 (discussed earlier) is correct, then the methyl transfer from the methylated corrinoid protein to CODH should be crippled. However, this reaction occurred at equal rates with the wild-type protein and the CFeSP variant. We feel that this result rules out the possibility of a radical methyl transfer mechanics and offers strong support for mechanism 1. 

An examination of mutant PKA proteins was undertaken. Phosphorylation of Thr-197 is required to activate PKA and phosphorylation of Ser-338 enhances stability of the protein. Replacement of Thr-197 and/or Ser-338 by Ala was examined to determine any conformational changes in the protein. Both single substitution mutants were expressed in Escherichia coli in similar levels to wild-type protein. However, both mutants were found to be less stable, as had been previously described. The double mutant... 

GPCR and transporter protein information from SWISS-PROT has been imported as background data into the Arcadia database. This background information is used in Arcadia to simplify mutant submissions (wild-type proteins are already in the database and therefore do not have to be added by the user residue positions of the mutations... 

It is also essential that any functional properties of the mutant protein that can be assessed be assessed. Although the substitution of one particular residue for another may be made in an attempt to determine the effect of the mutation on a specific property of a protein, it is quite possible that other properties that are not of immediate concern may be modified unintentionally and that these modifications may have important, otherwise occult, implications for the functional studies that are of immediate interest (vide infra). In the case of electron transfer proteins it may be useful, for example, to produce a family of mutants the members of which differ from each other only in their reduction potentials. This result may prove to be difficult to achieve because many mutations that perturb the reduction potential of a protein may also change its electrostatic properties or its reorganizational barrier to electron transfer. Depending on the experiments to be conducted with the mutants, these other properties may prove to be more important considerations than the reduction potentials of the mutants. In summary, new mutant proteins are ideally studied as if they were altogether new proteins of the same general class as the wild-type protein, and assumptions regarding the properties of such mutants should be kept to a minimum. 

In addition to the effect of mutations at Phe-82 on the stability of the cytochrome c active site, the intense, negative Soret Cotton effect in the circular dichroism spectrum of ferricytochrome c is profoundly affected by the presence of non-aromatic amino acid residues at this position [115]. Recent examination of six position-82 iso-l-ferricytochrome c mutants establishes that while Tyr-82 exhibits a Soret CD spectrum closely similar to that of the wild-type protein, the intensity of the negative Soret Cotton affect varies with the identity of the residue at this position in the order Phe > Tyr > Gly > Ser = Ala > Leu > He, though the Ser, Ala, He, and Leu variants have effectively no negative Soret Cotton effect [108]. 

Fig. 2a-c. Stereodiagram of the yeast iso-1-cytochrome c surface, (a) Surface of the wild-type protein (b) surface of the Ser-82 mutant (c) surface of the Gly-82 mutant. (Modified from Refs. [123, 124])... 

---