Protein mutagenesis

The importance of the interaction with the 2 -methyl group was revealed by a combination of protein mutagenesis and inhibitor modification (11). Site-directed mutants of COX-2 were generated, which altered the size of the hydrophobic pocket via... 

All these bioelectrocatalytic functions of redox proteins are based on the control and enhancement of the electrical communication between the redox sites of the proteins and the electrode support. This is accomplished by the nano-engineering of the surfaces with covalently anchored proteins, the structural aligmnent of the proteins on the electrodes and the chemical modification of the proteins with redox-active units. Preliminary results suggest that two approaches will play important roles in the future development of bioelectronic systems (i) protein mutagenesis with specific functional amino acid residues that can align the protein on the electrode surface and control the electrical contact with the electrode (ii) the synthesis of de novo proteins with tailored bioelectronic and electrobiocatalytic functions. 

McCammon JA, Gehn B, Karplus M (1977) Dynamics of folded proteins. Nature 267 585 Lopes A, Aleksandrov A, Bathelt C, Archontis G, Simonson T (2007) Computational sidechain placement and protein mutagenesis with implicit solvent models. Proteins 67 853-867... 

Dong SL, Ybe JA, Hecht MH, Spiro TG (1999) H-bonding maintains the active site of type 1 copper proteins mutagenesis of Asn38 in poplar plastocyanin. Biochemistry 38 3379-3385... 

Figure 16. General models for Hg(II) binding to the MerR protein. Mutagenesis studies led to the proposal of model 1 having linear bis coordination with ancillary ligation (173). Studies by electronic spectroscopy and Hg-EXAFS, indicate that Hg(II) is bound by three primary thiolate ligands, consistent with models 2 and 3, above (202, 209). Mutant complementation studies with Bacillus MerR support the model for Hg(II) bound by three primary bonds (83).

Stability and Purification of the Recombinant Protein. There are no hard and fast rules specifying, eg, whether a recombinant protein is available in a soluble state in the cell. In some cases, the expression system must be engineered by in vitro mutagenesis to optimize overall yield of the protein. 

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... 

Natural selection works through the complementary processes of mutation and genetic reassortment by recombination. The oligonucleotide-directed mutagenesis methods used in the foregoing examples do not allow for recombination instead, mutations are combined manually to optimize a protein sequence. Willem Stemmer at Maxygen invented a method of directed evolution that uses both mutation and recombination. This method, called... 

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. 

---