Proteins site-directed mutagenesis

Labelling Na,K-ATPase with ATP analogues provides evidence for contribution from charged residues that are widely separated in the sequence of a subunit of Na,K-ATPase. The first indication came from ATP sensitive covalent insertion of fluorescein-isothiocyanate (FITC) into Lys ° in the a subunit [90], The strong fluorescence signal provides a convenient probe for monitoring conformational transitions in the proteins. Site-directed mutagenesis of Lys reduces the activity of... 

Thioredoxin behaves differently from other proteins. Because of the great interest of the sulphur function in this enzyme (it is the active site of this thiol-disulfide oxidoreductase), the reduction of oxidized thioredoxin was studied in detail (139, 140). The disulfide radical is much more acidic than in other proteins, and its decay leads only to the reduced protein. Site-directed mutagenesis was used to modify selectively two amino-acids, Asp30 and Trp35, in order to observe the modulation of the redox properties of the sulphur functions. It was thus shown that both residues play a role in the proton transfer associated to electron transfer, although differently for instance, removal of W35 increases the pKa of... 

Cyt b559 is required for assembly of functional PSII. Deletion of the psbE or psbF genes in Synechocystis sp. PCC 6803 leads to the loss of PSII RC activity. Additionally, these mutants do not accumulate significant amount of the D1 and D2 proteins. Site-directed mutagenesis experiments that alter the putative heme ligands His-22 in either the a or P subunits also lead to dramatic losses of Dl, D2 and of both subunits of the cytochrome. Truncation of the carboxyl terminus of the a subunit by 31 amino acid residues leads to an 80-90% loss of PSII centers without the loss of assembled Cyt b559. This result indicates that the C-terminus of the subunit is not required for the formation of functional cytochrome but is required for the assembly of functional and stable PSII centers. 

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... 

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. 

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

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... 

The nonstructural region of the precursor, harboring the viral replication machinery, is cut into its mature components in a maturation reaction in which two viral proteases (NS2-pro and NS3/4A-pro) cooperate. Site-directed mutagenesis of an other wise infectious cDNA has shown that both HCV-encoded proteases are necessary for viral infectivity, but most of the attention has so far been focused on one of them a member of the serine protease family (EC 3.4.21) located in the N-terminal region of the viral NS3 protein. 

The family of serine proteases has been subjected to intensive studies of site-directed mutagenesis. These experiments provide unique information about the contributions of individual amino acids to kcat and KM. Some of the clearest conclusions have emerged from studies in subtilisin (Ref. 9), where the oxyanion intermediate is stabilized by t>e main-chain hydrogen bond of Ser 221 and an hydrogen bond from Asn 155 (Ref. 2). Replacement of Asn 155 (e.g., the Asn 155— Ala 155 described in Fig. 7.9) allows for a quantitative assessment of the effect of the protein dipoles on Ag. ... 

The coordination of the Rieske cluster in the a subunit of benzene dioxygenase has been studied by site-directed mutagenesis. The replacement of His 98 or His 119 (corresponding to His 83/104 in NDO) by Cys resulted in a protein that was unable to coordinate a normal Rieske-type cluster (53). In the mutant His 98 Cys, a novel EPR spectrum with av = 1-94 was detected that is intermediate between... 

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.

The Fe proteins are homodimers containing a single Fe4S4 cluster. Site-directed mutagenesis experiments showed that the cluster was probably held between the two subunits by ligation to two of the invariant cysteine residues from each subunit (16). This observation was confirmed later by X-ray crystallography (1) of the Fe protein... 

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