Site-directed mutagenesis of photoprotein mnemiopsin: implication of some conserved residues in bioluminescence properties

2013 ◽  
Vol 12 (3) ◽  
pp. 467-478 ◽  
Author(s):  
Atiyeh Mahdavi ◽  
Reza H. Sajedi ◽  
Saman Hosseinkhani ◽  
Majid Taghdir ◽  
Reyhaneh Sariri
Keyword(s):  
Site Directed Mutagenesis ◽  
Directed Mutagenesis ◽  
Conserved Residues

scholarly journals Conserved Residues Control the T1R3-Specific Allosteric Signaling Pathway of the Mammalian Sweet-Taste Receptor

2019 ◽  
Vol 44 (5) ◽  
pp. 303-310 ◽  
Author(s):  
Jean-Baptiste Chéron ◽  
Amanda Soohoo ◽  
Yi Wang ◽  
Jérôme Golebiowski ◽  
Serge Antonczak ◽  
...  
Keyword(s):  
Transmembrane Domain ◽  
Taste Receptor ◽  
Receptor Activation ◽  
Sweet Taste ◽  
Site Directed Mutagenesis ◽  
Activation Mechanism ◽  
Structural Basis ◽  
Directed Mutagenesis ◽  
Conserved Residues ◽  
Sweet Taste Receptor

Abstract Mammalian sensory systems detect sweet taste through the activation of a single heteromeric T1R2/T1R3 receptor belonging to class C G-protein-coupled receptors. Allosteric ligands are known to interact within the transmembrane domain, yet a complete view of receptor activation remains elusive. By combining site-directed mutagenesis with computational modeling, we investigate the structure and dynamics of the allosteric binding pocket of the T1R3 sweet-taste receptor in its apo form, and in the presence of an allosteric ligand, cyclamate. A novel positively charged residue at the extracellular loop 2 is shown to interact with the ligand. Molecular dynamics simulations capture significant differences in the behavior of a network of conserved residues with and without cyclamate, although they do not directly interact with the allosteric ligand. Structural models show that they adopt alternate conformations, associated with a conformational change in the transmembrane region. Site-directed mutagenesis confirms that these residues are unequivocally involved in the receptor function and the allosteric signaling mechanism of the sweet-taste receptor. Similar to a large portion of the transmembrane domain, they are highly conserved among mammals, suggesting an activation mechanism that is evolutionarily conserved. This work provides a structural basis for describing the dynamics of the receptor, and for the rational design of new sweet-taste modulators.

scholarly journals The PDZ Domain of the SpoIVB Serine Peptidase Facilitates Multiple Functions

2001 ◽  
Vol 183 (14) ◽  
pp. 4364-4373 ◽  
Author(s):  
Ngo T. Hoa ◽  
James A. Brannigan ◽  
Simon M. Cutting
Keyword(s):  
Gene Expression ◽  
Pdz Domain ◽  
Proteolytic Processing ◽  
Site Directed Mutagenesis ◽  
Directed Mutagenesis ◽  
Critical Component ◽  
Conserved Residues ◽  
Multiple Functions ◽  
Serine Peptidase ◽  
Distinct Process

ABSTRACT During spore formation in Bacillus subtilis, the SpoIVB protein is a critical component of the ςKregulatory checkpoint. SpoIVB has been shown to be a serine peptidase that is synthesized in the spore chamber and which self-cleaves, releasing active forms. These forms can signal proteolytic processing of the transcription factor ςK in the outer mother cell chamber of the sporulating cell. This forms the basis of the ςK checkpoint and ensures accurate ςK-controlled gene expression. SpoIVB has also been shown to activate a second distinct process, termed the second function, which is essential for the formation of heat-resistant spores. In addition to the serine peptidase domain, SpoIVB contains a PDZ domain. We have altered a number of conserved residues in the PDZ domain by site-directed mutagenesis and assayed the sporulation phenotype and signaling properties of mutant SpoIVB proteins. Our work has revealed that the SpoIVB PDZ domain could be used for up to four distinct processes, (i) targeting of itself for transproteolysis, (ii) binding to the protease inhibitor BofC, (iii) signaling of pro-ςK processing, and (iv) signaling of the second function of SpoIVB.

scholarly journals Phylogenetic analysis of the phytochrome superfamily reveals distinct microbial subfamilies of photoreceptors

2005 ◽  
Vol 392 (1) ◽  
pp. 103-116 ◽  
Author(s):  
Baruch Karniol ◽  
Jeremiah R. Wagner ◽  
Joseph M. Walker ◽  
Richard D. Vierstra
Keyword(s):  
Red Light ◽  
Site Directed Mutagenesis ◽  
Side Chain ◽  
Directed Mutagenesis ◽  
Conserved Residues ◽  
Genomic Analyses ◽  
Biochemical Studies ◽  
Lyase Activity ◽  
Acidic Environments ◽  
Micro Organisms

Phys (phytochromes) are a superfamily of photochromic photoreceptors that employ a bilin-type chromophore to sense red and far-red light. Although originally thought to be restricted to plants, accumulating genetic and genomic analyses now indicate that they are also prevalent among micro-organisms. By a combination of phylogenetic and biochemical studies, we have expanded the Phy superfamily and organized its members into distinct functional clades which include the phys (plant Phys), BphPs (bacteriophytochromes), Cphs (cyanobacterial Phys), Fphs (fungal Phys) and a collection of Phy-like sequences. All contain a signature GAF (cGMP phosphodiesterase/adenylate cyclase/FhlA) domain, which houses the bilin lyase activity. A PHY domain (uppercase letters are used to denote the PHY domain specifically), which helps stabilize the Pfr form (far-red-light-absorbing form of Phy), is downstream of the GAF region in all but the Phy-like sequences. The phy, Cph, BphP and Fph families also include a PLD [N-terminal PAS (Per/Arnt/Sim)-like domain] upstream of the GAF domain. Site-directed mutagenesis of conserved residues within the GAF and PLD motifs supports their importance in chromophore binding and/or spectral activity. In agreement with Lamparter, Carrascal, Michael, Martinez, Rottwinkel and Abian [(2004) Biochemistry 43, 3659–3669], a conserved cysteine within the PLD of several BphPs was found to be necessary for binding the chromophore via the C-3 vinyl side chain on the bilin A ring. Phy-type sequences were also discovered in the actinobacterium Kineococcus radiotolerans and collections of microorganisms obtained from marine and extremely acidic environments, thus expanding further the range of these photoreceptors. Based on their organization and distribution, the evolution of the Phy superfamily into distinct photoreceptor types is proposed.

Site-Directed Mutagenesis of the Conserved Residues in Component I ofBacillus subtilisHeptaprenyl Diphosphate Synthase†

Biochemistry ◽  
1999 ◽  
Vol 38 (44) ◽  
pp. 14638-14643 ◽  
Author(s):  
Yuan-Wei Zhang ◽  
Xiao-Yuan Li ◽  
Hiroshi Sugawara ◽  
Tanetoshi Koyama
Keyword(s):  
Site Directed Mutagenesis ◽  
Directed Mutagenesis ◽  
Conserved Residues

scholarly journals Role of cationic amino acids in the Na+/dicarboxylate co-transporter NaDC-1

2000 ◽  
Vol 350 (3) ◽  
pp. 677-683 ◽  
Author(s):  
Ana M. PAJOR ◽  
Esther S. KAHN ◽  
Rama GANGULA
Keyword(s):  
Amino Acids ◽  
Plasma Membrane ◽  
Xenopus Oocytes ◽  
Site Directed Mutagenesis ◽  
Directed Mutagenesis ◽  
Wild Type ◽  
Conserved Residues ◽  
In The Wild ◽  
Cationic Amino Acids

The role of cationic amino acids in the Na+/dicarboxylate co-transporter NaDC-1 was investigated by site-directed mutagenesis and subsequent expression of mutant transporters in Xenopus oocytes. Of the ten residues chosen for mutagenesis, eight (Lys-34, Lys-107, Arg-108, Lys-333, Lys-390, Arg-368, Lys-414 and Arg-541) were found to be non-essential for function or targeting. Only two conserved residues, Lys-84 (at the cytoplasmic end of helix 3) and Arg-349 (at the extracellular end of helix 7), were found to be important for transport. Both mutant transporters were expressed at the plasma membrane. The mutation of Lys-84 to Ala resulted in an increased Km for succinate of 1.8mM, compared with 0.3mM in the wild-type NaDC-1. The R349A mutant had Na+ and citrate kinetics that were similar to those of the wild type. However, succinate handling in the R349A mutant was altered, with evidence of inhibition at high succinate concentrations. In conclusion, charge neutralization of Lys-84 and Arg-349 in NaDC-1 affects succinate handling, suggesting that these residues might have roles in substrate binding.

Sign in / Sign up

Export Citation Format

Share Document