Site-directed mutagenesis experiments

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... 

Residue 189 is at the bottom of the specificity pocket. In trypsin the Asp residue at this position interacts with the positively charged side chains Lys or Arg of a substrate. This accounts for the preference of trypsin to cleave adjacent to these residues. In chymotrypsin there is a Ser residue at position 189, which does not interfere with the binding of the substrate. Bulky aromatic groups are therefore preferred by chymotrypsin since such side chains fill up the mainly hydrophobic specificity pocket. It has now become clear, however, from site-directed mutagenesis experiments that this simple picture does not tell the whole story. 

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. 

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... 

Fig. 2.5. Possible applications of a coupling parameter, A, in free energy calculations, (a) and (b) correspond, respectively, to simple and coupled modifications of torsional degrees of freedom, involved in the study of conformational equilibria (c) represents an intramolecular, end-to-end reaction coordinate that may be used, for instance, to model the folding of a short peptide (d) symbolizes the alteration of selected nonbonded interactions to estimate relative free energies, in the spirit of site-directed mutagenesis experiments (e) is a simple distance separating chemical species that can be employed in potential of mean force (PMF) calculations and (f) corresponds to the annihilation of selected nonbonded interactions for the estimation of e.g., free energies of solvation. In the examples (a), (b), and (e), the coupling parameter, A, is not independent of the Cartesian coordinates, x. Appropriate metric tensor correction should be considered through a relevant transformation into generalized coordinates...

Amino acid-sequence alignments of various LSs and ISs, in combination with site-directed mutagenesis experiments and structural data of family members, have highlighted functional amino acid residues, and has provided first insights into... 

Kristiansen, K., Dahl, S. G. and Edvardsen, O. A database of mutants and effects of site-directed mutagenesis experiments on G protein-coupled receptors. Proteins 26 81-94, 1996. 

A detailed model for the enzyme mechanism has been developed by Beinert and Kennedy and their collaborators on the basis of extensive spectroscopic studies, isotope labeling, kinetic experiments, several crystal structures, and site-directed mutagenesis experiments. As usual for an enzymological work in progress, the hardness of individual aspects of the model ranges from well established to conjectural. 

Preliminary site-directed mutagenesis experiments of the fused 1,2-a-D-mannosidase gene (f-msdS) on the expression vector, pGAM-1, were done previously to identify the functional role of catalytic residues in the 1,2-a-D-mannosidase from A. saitoi expressed in yeast cells [113],... 

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