Site-directed mutagenesis complex

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... 

In the three-dimensional stmcture of actin, the environment of the phosphate moiety of the nucleotide appears roughly the same when CaADP or CaATP is bound. This observation argues against two different conformations. The reason why this is so is unclear. However, it must be stressed that the three-dimensional stmcture is derived from X-ray diffraction of crystals of the DNasel-actin complex, which, like G-actin, is unable to hydrolyze ATP. The conformation obtained may therefore correspond to G-actin frozen in the G-ATP state independently of the bound nucleotide. Stmctural studies in conjunction with site-directed mutagenesis experiments should eventually solve this problem. 

Fig. 14. Plot of the g values g,g ) and of the average g value g vs rhombicity (UJ of (a) wild type (open symbol) and variant forms (closed symbols) of the Rieske protein in yeast bci complex where the residues Ser 183 and Tyr 185 forming hydrogen bonds into the cluster have been replaced by site-directed mutagenesis [Denke et al. (35) Merbitz-Zahradnik, T. Link, T. A., manuscript in preparation] and of (b) the Rieske cluster in membranes of Rhodobacter capsulatus in different redox states of the quinone pool and with inhibitors added [data from Ding et al. (79)]. The solid lines represent linear fits to the data points the dashed lines reproduce the fits to the g values of all Rieske and Rieske-type proteins shown in Fig. 13.

One example of the combined use of site-directed mutagenesis with chemical modification has been provided by the work of Bowler et al. with yeast iso-l-cytochrome c [15]. These workers introduced a His residue at position 62 to provide a site for attachment of a pentammineruthenium complex. Introduction of a second redox-active metal center to the protein at this position permitted... 

As part of a subsequent study concerning primarily second-site revertant yeast iso-l-cytochrome c variants, Hazzard et al. evaluated the effect of converting Lys-72 to an aspartyl residue by site-directed mutagenesis on the electron transfer kinetics of the cytochrome c-cytochrome c peroxidase complex [136]. Lys-72 was of interest for this purpose, because it is involved in the hypothetical model for the complex formed by these two proteins that was proposed by Poulos and Kraut on the basis of molecular graphics docking [106]. In these... 

The reference 28 authors continue to detail experimental observations that place voltage sensor helices in positions within the membrane. Miller and coworkers conducted site-directed mutagenesis for all residues of helices Sl-S3. ° In these experiments, tryptophan (trp) residues were substituted for each amino acid in turn to determine which residues would be trp-tolerant. These experiments confirmed a-helical conformations for SI and S2 and showed that K+ channel function was altered when trp residues were placed in some (labeled non-trp-tolerant), but not all, positions. The same treatment for helix S3 yielded complex results. At S3 s N-terminal end the distribution of trp-tolerant positions were consistent with an a-helical structure, however, this was not the case at S3 s C-terminal end. Other tests indicated that S3 might be helical for its entire length and that the N-terminal end interfaces with both lipid and protein while the C-terminal end interfaces with water. Comparisons of trp-tolerant or trp-intolerant residues over several different Kv channel... 

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