Site-specific mutations

SITE-SPECIFIC MUTATIONS PROVIDE A POWERFUL WAY OF EXPLORING DIFFERENT CATALYTIC MECHANISMS... 

Site-specific mutations, see Mutations, site-specific... 

Subtilisin, 170 active site of, 171,173 autocorrelation function of, 216, 216 potential surfaces for, 218 site-specific mutations, 184, 185, 187-188 Sugars, see Oligosaccharides Surface-constrained solvent model, 125... 

Site-Specific Mutations Provide a Powerful Way of Exploring Different Catalytic Mechanisms, 184... 

Qiu WH, Kao YT, Zhang LY, Yang Y, Wang LJ, Stites WE, Zhong DP, Zewail AH (2006) Protein surface hydration mapped by site-specific mutations. Proc Natl Acad Sci USA 103 (38) 13979-13984... 

Reeves, C.D., Murli, S., Ashley, G.W. et al. (2001) Alteration of the substrate specificity of a modular polyketide synthase acyltransferase domain through site-specific mutations. Biochemistry, 40, 15464. 

Narhi et al. (1991) recently reported an enhancement in the thermal stability of aprA-subtilisin by three point mutations. The mutations were ASNi. SER and ASN. SER to prevent cyclisation with the adjacent glycines and ASN . ASP in the Ca binding loop. The mutant form also exhibits improved stability to detergent denaturation with little dependence on calcium concentration. Subtilisin 8350 (derived from subtilisin BPN via six site-specific mutations) was found to be 100 times more stable than the wild type enzyme in aqueous solution and 50 times more stable than the wild type in anhydrous dimethylformamide (Wong et al, 1990)... 

Wong, C.-H., Chen, S.-T., Heimen, W.J., Hennen, W.J., Bibbs, J.A., Yang, Y.-F. etal. (1990) Enzymes in organic synthesis use of snbtilisin and a highly stable mutant derived from multiple site-specific mutations. J. Am. Chem. Soc, 112, 945-953. Wutrich, K. (1986) NMR of Proteins and Nucleic Adds. New York Wiley. 

The importance of non-polar residues to binding of methotrexate has also been examined, both by site-specific mutation (Taira and Benkovic, 1988)... 

Palczewski, K. Ohguro, H. Premont, R.T. Inglese, J. Rhodopsin kinase autophosphorylation. Characterization of site-specific mutations. J. Biol. Chem., 270, 15294-15298 (1995)... 

Zhou, L. Thornburg, R. Site-specific mutations of conserved residues in the phosphate-binding loop of the Arabidopsis UMP/CMP kinase alter ATP and UMP binding. Arch. Biochem. Biophys., 358, 297-302 (1998)... 

Site specific mutations can also be achieved using recombinant techniques. This facilitates study of a protein s activity if given amino acids are replaced in its structure. Thus, the protein s binding properties with respect to inhibitors, where the inhibitors represent an experimental drug, can be studied,... 

Previously, AAT had been transformed into an L-tyrosine aminotransferase (TAT) by site-specific mutation of up to six amino acid residues lining the active site of wild-type AAT. The hextuple AAT-mutant achieved kinetic data towards the transamination of aromatic substrates such as i-phenylalanine within an order of magnitude of wild-type TAT (Onuffer, 1995). 

At this point, we had assembled a preliminary model of the human thrombin receptor that contained all three extracellular loops linked to the seven TM domains, as well as the N-terminus out to residue 75. Docking studies with agonist peptide SFLLRN, and additional mutagenesis experiments, were carried out in parallel. Measurement of the effects of site-specific mutations in human PAR-1 identified several key amino acids of the receptor as being important for activation (Table 1),... 

Each one of the helices in bR manifested certain intrinsic stability towards the overall stability of the transmembrane (TM) helix. The helices A,B,C,D,E,F,G p0ssess an unique intrinsic potential barrier against unfolding or denaturation. The forces exerted on individual helices vary with temperature and from the force profiles of individual chains, the energy landscape of bR can be mapped. Enhancement of thermal stability of bR16,17 by site-specific mutations has occupied the attention of our laboratory for many years. 

Hypotheses regarding the consequences of specific amino acid replacements on substrate specificity can now be tested experimentally through site-directed mutagenesis of carboxypeptidase.25 Site-specific mutations can be introduced into cloned DNA by several highly efficient and simple methods. These methods have been summarized26 and are not detailed here. 

These studies have been essentially devoted to Cu2,Zn2SOD. The first NMRD profiles were reported for the bovine isoenzyme they showed a peculiar dispersion that is quite temperature dependent (Gaber et al., 1972). After development of the appropriate theory (Bertini etal., 1985a, 1985b, 1988 Koenig and Brown, 1987), the fitting of the NMRD profiles indicated the presence of one water molecule with the protons ca. 3.4 A from the copper ion. The latter is characterized by an electron relaxation time of 2.2 x 10 s at 298 K (Bertini et al., 1988). Typical prohles are reported in Fig. 9. Afterward, this technique was extensively used to characterize the presence or absence of the water molecule close to the copper ion under a variety of experimental conditions, such as in the presence of anions and site-specific mutations (Bertini et al., 1998, and references therein). When anions that act as strong inhibitors and... 

Reeves CD, Murli S, Ashley GW, Piagentini M, Hutchinson CR, McDaniel R (2001) Alteration of the Substrate Specificity of a Modular Polyketide Synthase Acyltransferase Domain Through Site-Specific Mutations. Biochemistry 40 15464... 

Thirty years of research with bacteriorhodopsin has provided answers to many questions about how protons are transported by transmembrane pumps. In this small seven-transmembrane protein, absorption of light by the retinal chromophore Initiates a reaction cycle in which the initial state recovers through multiple conformational changes of the retinal and the protein, and a proton Is translocated stepwise from one side of the membrane to the other. Spectroscopy, extensive use of site-specific mutations, and crystallography have defined the photocycle reactions in atomic detail and provide a step-by-step description of the proton transfers, the transient local and global perturbations in the protein and how they arise, and the energy flow through the system, which add up to the mechanism of the pump. 

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