Scanning mutagenesis of regions in the Galpha protein Gpa1 that are predicted to interact with yeast mating pheromone receptors.

The mechanism by which receptors activate heterotrimeric G proteins was examined by scanning mutagenesis of the Saccharomyces cerevisiae pheromone-responsive Galpha protein (Gpa1). The juxtaposition of high-resolution structures for rhodopsin and its cognate G protein transducin predicted that at least six regions of Galpha are in close proximity to the receptor. Mutagenesis was targeted to residues in these domains in Gpa1, which included four loop regions (beta2-beta3, alpha2-beta4, alpha3-beta5, and alpha4-beta6) as well as the N and C termini. The mutants displayed a range of phenotypes from nonsignaling to constitutive activation of the pheromone pathway. The constitutive activity of some mutants could be explained by decreased production of Gpa1, which permits unregulated signaling by Gbetagamma. However, the constitutive activity caused by the F344C and E335C mutations in the alpha2-beta4 loop and F378C in the alpha3-beta5 loop was not due to decreased protein levels, and was apparently due to defects in sequestering Gbetagamma. The strongest loss of the function mutant, which was not detectably induced by a pheromone, was caused by a K314C substitution in the beta2-beta3 loop. Several other mutations caused weak signaling phenotypes. Altogether, these results suggest that residues in different interface regions of Galpha contribute to activation of signaling.     

A systematic and comprehensive combinatorial approach to simultaneously improve the activity, reaction specificity, and thermal stability of p-hydroxybenzoate hydroxylase

We have simultaneously improved the activity, reaction specificity, and thermal stability of p-hydroxybenzoate hydroxylase by means of systematic and comprehensive combinatorial mutagenesis starting from available single mutations. Introduction of random mutations at the positions of four cysteine and eight methionine residues provided 216 single mutants as stably expressed forms in Escherichia coli host cells. Four characteristics, hydroxylase activity toward p-hydroxybenzoate (main activity), protocatechuate-dependent NADPH oxidase activity (sub-activity), ratio of sub-activity to main activity (reaction specificity), and thermal stability, of the purified mutants were determined. To improve the above characteristics for diagnostic use of the enzyme, 11 single mutations (C152V, C211I, C332A, M52V, M52Q, M110L, M110I, M213G, M213L, M276Q, and M349A) were selected for further combinatorial mutagenesis. All possible combinations of the mutations provided 18 variants with double mutations and further combinatorial mutagenesis provided 6 variants with triple mutations and 9 variants with quadruple mutations with the simultaneously improved four properties.     

Clustered charged-to-alanine mutagenesis of poliovirus RNA-dependent RNA polymerase yields multiple temperature-sensitive mutants defective in RNA synthesis.

To generate a collection of conditionally defective poliovirus mutants, clustered charged-to-alanine mutagenesis of the RNA-dependent RNA polymerase 3D was performed. Clusters of charged residues in the polymerase coding region were replaced with alanines by deoxyoligonucleotide-directed mutagenesis of a full-length poliovirus cDNA clone. Following transfection of 27 mutagenized cDNA clones, 10 (37%) gave rise to viruses with temperature-sensitive (ts) phenotypes. Three of the ts mutants displayed severe ts plaque reduction phenotypes, producing at least 10(3)-fold fewer plaques at 39.5 degrees C than at 32.5 degrees C; the other seven mutants displayed ts small-plaque phenotypes. Constant-temperature, single-cycle infections showed defects in virus yield or RNA accumulation at the nonpermissive temperature for eight stable ts mutants. In temperature shift experiments, seven of the ts mutants showed reduced accumulation of viral RNA at the nonpermissive temperature and showed no other ts defects. The mutations responsible for the phenotypes of most of these ts mutants lie in the N-terminal third of the 3D coding region, where no well-characterized mutations responsible for viable mutants had been previously identified. Clustered charged-to-alanine mutagenesis (S. H. Bass, M. G. Mulkerrin, and J. A. Wells, Proc. Natl. Acad. Sci. USA 88:4498-4502, 1991; W. F. Bennett, N. F. Paoni, B. A. Keyt, D. Botstein, J. J. S. Jones, L. Presta, F. M. Wurm, and M. J. Zoller, J. Biol. Chem. 266:5191-5201, 1991; and K. F. Wertman, D. G. Drubin, and D. Botstein, Genetics 132:337-350, 1992) is designed to target residues on the surfaces of folded proteins; thus, extragenic suppression analysis of such mutant viruses may be very useful in identifying components of the viral replication complex.     

Combinatorial mutagenesis of the lamB gene: residues 41 through 43, which are conserved in Escherichia coli outer membrane proteins, are informationally important in maltoporin structure and function.

A new strategy for combinatorial mutagenesis was developed and applied to residues 40 through 60 of LamB protein (maltoporin), with the aim of identifying amino acids important for LamB structure and function. The strategy involved a template containing a stop codon in the target sequence and a pool of random degenerate oligonucleotides covering the region. In vitro mutagenesis followed by selection for function (Dex+, ability to utilize dextrins) corrected the nonsense mutation and simultaneously forced incorporation of a random mutation(s) within the region. The relative importance of each residue within the target was indicated by the frequency and nature of neutral and deleterious mutations recovered at each position. Residues 41 through 43 in LamB accepted few neutral substitutions, whereas residues 55 through 57 were highly flexible in this regard. Consistent with this finding was that the majority of defective mutants were altered at residues 41 to 43. Characterization of these mutants indicated that the nature of residues 41 to 43 influenced the amount of stable protein in the outer membrane. These results, as well as the conserved nature of this stretch of residues among outer membrane proteins, suggest that residues 41 to 43 of LamB play an important role in the process of outer membrane localization.     

Phenotypes of combined tet repressor mutants for effector and operator recognition and allostery

Tet repressor mutants with a shifted effector specificity, preference for a mutant operator sequence or reversion of activity were combined to construct variants bearing two or three phenotypic alterations. TetR alleles with combinations of altered operator and effector specificities can be created by merging the respective residues in a single polypeptide. The mutations giving rise to revTetR, on the other hand, show drastic influences on the ligand binding phenotypes when combined with respective alterations. One TetR variant displays all three phenotypic alterations and thus demonstrates the general possibility of implementing them in one protein.