Search results for "site-directed mutagenesis"

Effect of substitutions of key residues on the stability and the insecticidal activity of Vip3Af from Bacillus thuringiensis

2021

Modern agriculture demands for more sustainable agrochemicals to reduce the environmental and health impact. The whole process of the discovery and development of new active substances or control agents is sorely slow and expensive. Vegetative insecticidal proteins (Vip3) from Bacillus thuringiensis are specific toxins against caterpillars with a potential capacity to broaden the range of target pests. Site-directed mutagenesis is one of the most approaches used to test hypotheses on the role of different amino acids on the structure and function of proteins. To gain a better understanding of the role of key amino acid residues of Vip3A proteins, we have generated 12 mutants of the Vip3Af1 …

Evaluation of an amino acid residue critical for the specificity and activity of human Gb3/CD77 synthase

2016

Human Gb3/CD77 synthase (α1,4-galactosyltransferase) is the only known glycosyltransferase that changes acceptor specificity because of a point mutation. The enzyme, encoded by A4GALT locus, is responsible for biosynthesis of Gal(α1–4)Gal moiety in Gb3 (CD77, Pk antigen) and P1 glycosphingolipids. We showed before that a single nucleotide substitution c.631C > G in the open reading frame of A4GALT, resulting in replacement of glutamine with glutamic acid at position 211 (substitution p. Q211E), broadens the enzyme acceptor specificity, so it can not only attach galactose to another galactose but also to N-acetylgalactosamine. The latter reaction leads to synthesis of NOR antigens, which are…

Biophysical and functional characterization of the human olfactory receptor OR1A1 expressed in a mammalian inducible cell line

2014

International audience; Olfactory receptors (ORs) play a crucial role in detecting the odorant molecules present in the surrounding environment. These receptors, which belong to class A G-protein-coupled receptors, constitute the largest transmembrane protein family in the human genome. Functional studies showed that the OR family includes members that are able to respond to a large set of odorants and members that are activated by a relatively small number of related odorants. To understand the molecular mechanisms that govern the receptor-ligand interactions, we overexpressed the human OR hOR1A1 in a stable tetracycline-inducible HEK293S cell line. This receptor was engineered by insertin…

2016

Urm1 is a unique dual-function member of the ubiquitin protein family and conserved from yeast to man. It acts both as a protein modifier in ubiquitin-like urmylation and as a sulfur donor for tRNA thiolation, which in concert with the Elongator pathway forms 5-methoxy-carbonyl-methyl-2-thio (mcm5s2) modified wobble uridines (U34) in anticodons. Using Saccharomyces cerevisiae as a model to study a relationship between these two functions, we examined whether cultivation temperature and sulfur supply previously implicated in the tRNA thiolation branch of the URM1 pathway also contribute to proper urmylation. Monitoring Urm1 conjugation, we found urmylation of the peroxiredoxin Ahp1 is suppre…

Are glutamate and asparagine necessary for tyrosinase activity of type-3 copper proteins?

2018

Abstract Type-3 copper proteins (T3CPs) are complex proteins which share similar active sites. Two copper atoms (CuA and CuB) bind dioxygen as a peroxide in a side on coordination. This protein family comprises the enzymes tyrosinase and catechol oxidase as well as the oxygen transporter hemocyanin. T3CPs occur in almost all organisms and exhibit a number of essential functions. In particular, they are involved in all kinds of enzymatic browning reactions and immune defense. The chemical basis of the two catalytic processes, i.e., the o-hydroxylation of monophenols and the two-electron oxidation to o-quinones, is still discussed. Investigations on natural enzymes with known crystal structur…

Site-directed mutagenesis of odorant-binding proteins

2020

Modifying the affinity of odorant-binding proteins (OBPs) to small ligands by replacement of specific residues in the binding pocket may lead to several technological applications. Thanks to their compact and stable structures, OBPs are currently regarded as the best candidates to be used in biosensing elements for odorants and volatiles detection. The wide and rich information on the structure of these proteins both in their apo-forms and in complexes with specific ligands provides guidelines to design reliable mutants to monitor specific targets. The same engineered proteins may also find applications in the slow release of pheromones and other chemicals in the environment, as well as in …

Structural and Functional Analysis of the Antiparallel Strands in the Lumenal Loop of the Major Light-harvesting Chlorophyll a/b Complex of Photosyst…

2007

The light-harvesting chlorophyll a/b-binding protein of photosystem II (LHCIIb) fulfills multiple functions, such as light harvesting and energy dissipation under different illuminations. The crystal structure of LHCIIb at the near atomic resolution reveals an antiparallel strands structure in the lumenal loop between the transmembrane helices B/C. To study the structural and functional significances of this structure, three amino acids (Val-119, His-120, and Ser-123) in this region have been exchanged to Phe, Leu, and Gly, respectively, and the influence of the mutagenesis on the structure and function of LHCIIb has been investigated. The results are as follows. 1) Circular dichroism spect…

Precise mapping of the Goodpasture epitope(s) using phage display, site-directed mutagenesis, and surface plasmon resonance.

2013

Goodpasture disease is an autoimmune disorder mediated by circulating autoantibodies against the noncollagenous-1 (NC1) domain of the alpha 3 chain of type IV collagen (alpha 3(IV)NC1). The structure of Goodpasture epitope(s) has been previously mapped into two main binding regions (E-A and E-B) of the alpha 3(IV)NC1 domain using a residue mutation approach on the highly related alpha 1(IV)NC1 domain. Here we combined phage display and surface plasmon resonance technology to more precisely localize the pathogenic binding sites. Peptides mimicking the Goodpasture epitope(s) were used to identify residues involved in autoantibody binding and found involvement of eight residues previously unre…

The Study of Carbamoyl Phosphate Synthetase 1 Deficiency Sheds Light on the Mechanism for Switching On/Off the Urea Cycle

2015

12 páginas, 4 figuras, 2 tablas.

Role of two operators in regulating the plasmid-borne raf operon of Escherichia coli

1994

The plasmid-borne raf operon encodes functions required for the inducible uptake and utilization of raffinose in Escherichia coli K12. The expression of three structural genes for alpha-galactosidase (rafA), Raf permease (rafB) and sucrose hydrolase (rafD) is negatively controlled by the binding of RafR repressor (rafR) to two operator sites, O1 and O2, that flank the -35 sequence of the raf promoter, PA. In vitro, O1 and O2 are occupied on increasing the concentration of RafR, without detectable preference for one site or the other or any indication of cooperative binding. Nucleotide substitutions at positions 3, 4 or 5 in an operator half-site prevented repressor binding, supporting a mod…