A computer modeling procedure for assessing the stereochemicalsuitability of pairs of residues in proteins as potential sitesfor introduction of cystine disulfide crosslinks has been developed.Residue pairs with C C distances of 6.5 Å andCbeta;Cß distances of 4.5 Å are chosenfor geometrical fixation of S atoms using the program MODIP.The stereochemistry of the modeled disulfides is evaluated usinglimits for the structural parameters of the various torsionangles and SS bond length in the disulfide bridge. Theability of the procedure to correctly model disulfides has beenchecked with examples of cystine peptides of known crystal structuresand 103 disulfide bridges from 25 available protein crystalstructures determined at 2 Å resolution. An analysis ofresults on three proteins with engineered disulfides, T4 lysozyme,dihydrofolate reductase and subtilisin, is presented. Two positionsfor the introduction of stereochemically optimaldisulfides are identified in subtilisin. 相似文献
Site-directed mutagenesis has been used to change the codonfor cysteine-107 of Saccharomyces cerevisiae iso-1-cytochromec to a threonine codon. The resulting protein is active in vivo,is methylated as in the wild-type protein and has optical propertiesindistinguishable from those of the wild-type protein. The threonine-107iso-1-cytochrome c demonstrated fully reversible electrochemicalbehaviour and a mid-point reduction potential of 272 mV versusNHE. In addition, this mutant does not demonstrate a tendencyto autoreduce or to dimerize as does the wild-type protein.These properties of the threonine-107 mutant establish thatit will provide a useful background in which to make subsequentmutations for mechanistic and physical studies of yeast iso-1-cytochromec. 相似文献
The appearance dynamics of spontaneous revertants in adenine- or leucine-auxotrophic haploid Saccharomyces cerevisiae strains has been studied using mathematical simulation methods. In the case of adenine auxotrophs an increase in the number of revertants with decreasing metabolite content is found to result mainly from increasing the rate of intragenic suppressor mutations. In the case of leucine auxotrophs revertants result from increasing the appearance of mutants formed at similar rates under different cultivation conditions. In the latter case the appearance of mutant colonies increases with decreasing size of colonies of the initial auxotrophic cells. The last can simulate so-called adaptive mutagenesis. The heterogeneity of revertants appearing in different time periods is described. 相似文献
Site directed mutagenesis was used to introduce two cysteine residues into the hydrophilic regions of the pea vichlin polypeptide previously expressed in Saccharomyces cerevisiae. The mutated polypeptide was expresed and the resultant protein was characterised by sodium dodecyl sulphatepolyacrylamide gel electrophoresis. The presence of both intra- and inter-polypeptide sulphydryl bonds was indicated by bands of different mobility from those of the native polypeptide. The association of mutated vicili polypeptides into disulphide-bonded aggregates within the yeast was not a random process, trimers being the major aggregate produced. When the mutated vicilin was extracted from yeast and purified, random aggregates of the vicilin polypeptide were formed. 相似文献
Tailor made : We report the rational biosynthesis of C15 hydroxylated non‐quinone geldanamycin analogues by site‐directed mutagenesis of the geldanamycin polyketide synthase (PKS), together with a combination of post‐PKS tailoring genes. Rational biosynthetic engineering allowed the generation of geldanamycin derivatives, such as DHQ3 illustrated in the figure, which had superior pharmacological properties in comparison to the parent compound.
Probing the sheet : The network of hydrogen bonds formed in the outer β sheet of the nicotinic acetylcholine receptor (nAChR; see figure) is fairly robust and tolerates single amide‐to‐ester mutations throughout. However, eliminating two proximal hydrogen bonds completely destroys receptor function; this adds further support to gating models that ascribe important roles to these β strands of the nAChR extracellular domain.