Site-directed mutagenesis crystallization

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. 

Since these proteins are difficult to crystallize and site-directed mutagenesis has certain limitations, PAL can find much use in exploring pathways, transducers, and effectors in signal transduction as well as some of their main structural features. 

These interactions, plus others, stabilize the P4-P6 domain s conformation. Site-directed mutagenesis and chemical protection studies indicate that G212 in the P4 helix interacts with residues in the A-rich bulge, and the crystal structure shows interactions with A184 (02 G212-N1 A184 = 2.8 A, for example). The same sorts of interactions are found between the GAAA... 

The mechanisms for the rednction of compounds I and II are less well nnderstood than the mechanism of compound I formation, although a number of suggestions have been presented in the literature (24). It is clear from site-directed mutagenesis studies that Arg38 and His42 are also important in the rednction steps, although their precise role has been difficnlt to define. A detailed mechanism for snbstrate oxidation by plant peroxidases has been proposed, based on data from the crystal... 

Site-directed mutagenesis and the crystal structure analysis of a proteasome-inhibitor complex identified the amino-terminal threonine (Thrl) of Thermoplasma P subunits as both, the catalytic nucleophile and the primary proton acceptor (Seemiiller et al. 1995 Lowe et al. 1995). 

The distance measured by EPR is the point dipole distance between the two paramagnetic centres. When applied to structures of spin-labelled biomolecules the desired distance is between sites on the biomolecules. A key question is the conformation and conformational flexibility of the spin label. X-ray crystal structures of four spin-labelled derivatives of T4 lysozyme have been reported.54 Preferred rotational conformations of the linkage between the cysteine introduced by site-directed mutagenesis and the spin label were observed. The electron density associated with the nitroxide ring in different mutants was inversely correlated with the mobility of the label observed in fluid solution EPR spectra. 

The preferred modem procedure for investigating an enzyme mechanism is to identify a protein in a genome sequence, clone and express it, solve the crystal and/or solution structures, guess the mechanism, and then confirm it by site-directed mutagenesis and kinetics. The mechanism of lysozyme almost illustrates that procedure. Although the classical protein chemistry on lysozyme predated the molecular genetics, the kinetic studies were stimulated by the x-ray crystallography, and the previously unknown mechanism almost leapt from the... 

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