Site-directed mutagenesis conformation

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. 

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. 

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... 

While the ddNs and ANPs must be converted intracellularly to their 5 -triphosphates (ddNTPs) or diphosphate derivatives before they can interact as competitive inhibitors/alternate substrates with regard to the natural substrates (dNTPs), the NNRTIs do not need any metabolic conversion to interact, noncompetitively with respect to the dNTPs, at an allosteric, non-substrate binding site of the HIV-1 RT. Through the analysis of NNRTI-resistant mutants, combined with site-directed mutagenesis studies, it has become increasingly clear which amino acid residues are involved in the interaction of the NNRTIs with HIV-1 RT, and, since the conformation of the HIV-1 RT has been resolved at 3.0 A resolution [73], it is now possible to visualize the binding site of the NNRTIs [74],... 

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...

Zecherle, G.N. (1990) The ribosomal location and conformation of Escherichia coli protein L7/L12 studied by cysteine site directed mutagenesis and crosslinking. Doctoral Dissertation. University of California at Davis. 

The reference 28 authors continue to detail experimental observations that place voltage sensor helices in positions within the membrane. Miller and coworkers conducted site-directed mutagenesis for all residues of helices Sl-S3. ° In these experiments, tryptophan (trp) residues were substituted for each amino acid in turn to determine which residues would be trp-tolerant. These experiments confirmed a-helical conformations for SI and S2 and showed that K+ channel function was altered when trp residues were placed in some (labeled non-trp-tolerant), but not all, positions. The same treatment for helix S3 yielded complex results. At S3 s N-terminal end the distribution of trp-tolerant positions were consistent with an a-helical structure, however, this was not the case at S3 s C-terminal end. Other tests indicated that S3 might be helical for its entire length and that the N-terminal end interfaces with both lipid and protein while the C-terminal end interfaces with water. Comparisons of trp-tolerant or trp-intolerant residues over several different Kv channel... 

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 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. 

CD in general is a useful tool for examining proteins made by site-directed mutagenesis for structure-function studies, where it is important to know to what extent the conformation has been... 

Efavirenz is a nonnucleoside reverse transcriptase inhibitor specific for HIV-1. After binding to a site distant from the active site on the HIV-1 reverse transcriptase, it disrupts catalytic activity of the enzyme by causing a conformational change and does not compete with deoxynucleoside triphosphates. Efavirenz does not inhibit HIV-2 reverse transcriptase and human DNA polymerases a, (3, 7 and 8. The resistance to the drug develops rapidly from site-directed mutagenesis specifically at codon 103, and also at codons 100, 106, 108, 181, 190 and 225 of viral reverse transcriptase. This resistance will be applicable for all nonnucleoside transcriptase inhibitors. 

---