Search results for " Site-Directed"
showing 10 items of 132 documents
Chicken Avidin-related Protein 4/5 Shows Superior Thermal Stability when Compared with Avidin while Retaining High Affinity to Biotin
2003
The protein chicken avidin is a commonly used tool in various applications. The avidin gene belongs to a gene family that also includes seven other members known as the avidin-related genes (AVR). We report here on the extremely high thermal stability and functional characteristics of avidin-related protein AVR4/5, a member of the avidin protein family. The thermal stability characteristics of AVR4/5 were examined using a differential scanning calorimeter, microparticle analysis, and a microplate assay. Its biotin-binding properties were studied using an isothermal calorimeter and IAsys optical biosensor. According to these analyses, in the absence of biotin AVR4/5 is clearly more stable (T…
Molecular Architecture of Strictosidine Glucosidase: The Gateway to the Biosynthesis of the Monoterpenoid Indole Alkaloid Family[W]
2007
Abstract Strictosidine β-d-glucosidase (SG) follows strictosidine synthase (STR1) in the production of the reactive intermediate required for the formation of the large family of monoterpenoid indole alkaloids in plants. This family is composed of ∼2000 structurally diverse compounds. SG plays an important role in the plant cell by activating the glucoside strictosidine and allowing it to enter the multiple indole alkaloid pathways. Here, we report detailed three-dimensional information describing both native SG and the complex of its inactive mutant Glu207Gln with the substrate strictosidine, thus providing a structural characterization of substrate binding and identifying the amino acids …
Nucleoside Analogue Mutagenesis of a Single-Stranded DNA Virus: Evolution and Resistance
2012
ABSTRACT It has been well established that chemical mutagenesis has adverse fitness effects in RNA viruses, often leading to population extinction. This is mainly a consequence of the high RNA virus spontaneous mutation rates, which situate them close to the extinction threshold. Single-stranded DNA viruses are the fastest-mutating DNA-based systems, with per-nucleotide mutation rates close to those of some RNA viruses, but chemical mutagenesis has been much less studied in this type of viruses. Here, we serially passaged bacteriophage ϕX174 in the presence of the nucleoside analogue 5-fluorouracil (5-FU). We found that 5-FU was unable to trigger population extinction for the range of conce…
Distribution of Fitness Effects Caused by Single-Nucleotide Substitutions in Bacteriophage f1
2010
Empirical knowledge of the fitness effects of mutations is important for understanding many evolutionary processes, yet this knowledge is often hampered by several sources of measurement error and bias. Most of these problems can be solved using site-directed mutagenesis to engineer single mutations, an approach particularly suited for viruses due to their small genomes. Here, we used this technique to measure the fitness effect of 100 single-nucleotide substitutions in the bacteriophage f1, a filamentous single-strand DNA virus. We found that approximately one-fifth of all mutations are lethal. Viable ones reduced fitness by 11% on average and were accurately described by a log-normal dist…
The fitness effects of synonymous mutations in DNA and RNA viruses.
2011
Despite being silent with respect to protein sequence, synonymous nucleotide substitutions can be targeted by natural selection directly at the DNA or RNA level. However, there has been no systematic assessment of how frequent this type of selection is. Here, we have constructed 53 single random synonymous substitution mutants of the bacteriophages Qb and UX174 by site-directed mutagenesis and assayed their fitness. Analysis of this mutant collection and of previous studies undertaken with a variety of single-stranded (ss) viruses demonstrates that selection at synonymous sites is stronger in RNA viruses than in DNA viruses. We estimate that this type of selection contributes approximately …
C172S Substitution in the Chloroplast-encoded Large Subunit Affects Stability and Stress-induced Turnover of Ribulose-1,5-bisphosphate Carboxylase/Ox…
1999
Previous work has indicated that the turnover of chloroplast ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco; EC 4.1.1. 39) may be controlled by the redox state of certain cysteine residues. To test this hypothesis, directed mutagenesis and chloroplast transformation were employed to create a C172S substitution in the Rubisco large subunit of the green alga Chlamydomonas reinhardtii. The C172S mutant strain was not substantially different from the wild type with respect to growth rate, and the purified mutant enzyme had a normal circular dichroism spectrum. However, the mutant enzyme was inactivated faster than the wild-type enzyme at 40 and 50 degrees C. In contrast, C172S mutant …
Cysteines 449 and 459 modulate the reduction-oxidation conformational changes of ribulose 1.5-bisphosphate carboxylase/oxygenase and the translocatio…
2006
The role of cysteines 449 (Cys449) and 459 (Cys459) from the large subunit (LS) of ribulose 1-5-bisphosphate carboxylase/oxygenase (Rubisco) in the reduction-oxidation (redox) regulation of the enzyme was assessed by site-directed mutagenesis of these residues and chloroplast transformation of Chlamydomonas reinhardtii. In vitro studies indicated that mutations C449S, C459S or C449S/ C459S do not affect the activity and proteolytic susceptibility of the enzyme in the reduced state. However, when oxidized, the mutant enzymes differed from the wild type (WT), showing an increased resistance to inactivation and, in the case of the double mutant (DM), an altered structural conformation as refle…
Single amino acids in the lumenal loop domain influence the stability of the major light-harvesting chlorophyll a/b complex.
2004
The major light-harvesting complex of photosystem II (LHCIIb) is one of the most abundant integral membrane proteins. It greatly enhances the efficiency of photosynthesis in green plants by binding a large number of accessory pigments that absorb light energy and conduct it toward the photosynthetic reaction centers. Most of these pigments are associated with the three transmembrane and one amphiphilic alpha helices of the protein. Less is known about the significance of the loop domains connecting the alpha helices for pigment binding. Therefore, we randomly exchanged single amino acids in the lumenal loop domain of the bacterially expressed apoprotein Lhcb1 and then reconstituted the muta…
The distribution of fitness effects caused by single-nucleotide substitutions in an RNA virus.
2004
6 pages, 3 figures.-- PMID: 15159545 [PubMed].-- PMCID: PMC420405.-- Supporting information (Table 3: Relevant information about each single-nucleotide substation mutant created) available at: http://www.pnas.org/content/101/22/8396/suppl/DC1
Inhibition of glycosaminoglycan modification of perlecan domain I by site-directed mutagenesis changes protease sensitivity and laminin-1 binding act…
1998
AbstractGlycosaminoglycan attachment to perlecan domain I (173 residues) was completely prevented by site-directed mutagenesis of Ser-65, Ser-71 and Ser-76 as shown by recombinant production in mammalian cells. This did not interfere with the proper folding of the domain's SEA module but enhanced its sensitivity to neutral proteases. Lack of substitution also abolished binding to the two major heparin binding sites of laminin-1.