Role of tryptophan 135 of Chandipura virus phosphoprotein P in dimerization and complex formation with leader RNA: structural aspect using time resolved anisotropy and simulation

RSC Advances ◽  
2015 ◽  
Vol 5 (126) ◽  
pp. 104582-104593
Author(s):  
Manini Mukherjee ◽  
Aditya Sarkar ◽  
Arunava Roy ◽  
Pinki Saha Sardar ◽  
Ansuman Lahiri ◽  
...  
Keyword(s):  
Molecular Dynamics ◽  
Structural Change ◽  
Complex Formation ◽  
Protein Concentration ◽  
Structural Aspect ◽  
Wild Type ◽  
Time Resolved ◽  
Type Protein ◽  
Wild Type Protein

The nanosecond and picosecond dynamics of wild type protein and its tryptophan mutants have been used to study structural change as a function of protein concentration and binding with leader RNA by time resolved anisotropy and molecular dynamics.

scholarly journals Expression of the Schwanniomyces occidentalis SWA2 amylase in Saccharomyces cerevisiae: role of N-glycosylation on activity, stability and secretion

1998 ◽  
Vol 329 (1) ◽  
pp. 65-71 ◽  
Author(s):  
Esther YÁÑEZ ◽  
A. Teresa CARMONA ◽  
Mercedes TIEMBLO ◽  
Antonio JIMÉNEZ ◽  
María FERNÁNDEZ-LOBATO
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Catalytic Efficiency ◽  
Extracellular Enzyme ◽  
Site Directed Mutagenesis ◽  
Activity Levels ◽  
Wild Type ◽  
Schwanniomyces Occidentalis ◽  
Type Protein ◽  
Wild Type Protein

The role of N-linked glycosylation on the biological activity of Schwanniomyces occidentalis SWA2 α-amylase, as expressed in Saccharomyces cerevisiae, was analysed by site-directed mutagenesis of the two potential N-glycosylation sites, Asn-134 and Asn-229. These residues were replaced by Ala or Gly individually or in various combinations and the effects on the activity, secretion and thermal stability of the enzyme were studied. Any Asn-229 substitution caused a drastic decrease in activity levels of the extracellular enzyme. In contrast, substitutions of Asn-134 had little or no effect. The use of antibodies showed that α-amylase was secreted in all the mutants tested, although those containing substitutions at Asn-229 seemed to have a lower rate of synthesis and/or higher degradation than the wild-type strain. α-Amylases with substitution at Asn-229 had a 2 kDa lower molecular mass than the wild-type protein, as did the wild-type protein itself after treatment with endoglycosidase F. These findings indicate that Asn-229 is the single glycosylated residue in SWA2. Thermostability analysis of both purified wild-type (T50 = 50 °C, where T50 is the temperature resulting in 50% loss of activity) and mutant enzymes indicated that removal of carbohydrate from the 229 position results in a decrease of approx. 3 °C in the T50 of the enzyme. The Gly-229 mutation does not change the apparent affinity of the enzyme for starch (Km) but decreases to 1/22 its apparent catalytic efficiency (kcat/Km). These results therefore indicate that glycosylation at the 229 position has an important role in the extracellular activity levels, kinetics and stability of the Sw. occidentalis SWA2 α-amylase in both its wild-type and mutant forms.

scholarly journals Importance of the two tryptophan residues in the Streptomyces R61 exocellular dd-peptidase

1992 ◽  
Vol 282 (2) ◽  
pp. 361-367 ◽  
Author(s):  
C Bourguignon-Bellefroid ◽  
J M Wilkin ◽  
B Joris ◽  
R T Aplin ◽  
C Houssier ◽  
...  
Keyword(s):  
Enzyme Activity ◽  
Enzymic Activity ◽  
Site Directed Mutagenesis ◽  
Wild Type ◽  
Beta Lactams ◽  
Beta Lactamases ◽  
Type Protein ◽  
Wild Type Protein ◽  
Tryptophan Residues

Modification of the Streptomyces R61 DD-peptidase by N-bromosuccinimide resulted in a rapid loss of enzyme activity. In consequence, the role of the enzyme's two tryptophan residues was investigated by site-directed mutagenesis. Trp271 was replaced by Leu. The modification yielded a stable enzyme whose structural and catalytic properties were similar to those of the wild-type protein. Thus the Trp271 residue, though almost invariant among the beta-lactamases of classes A and C and the low-Mr penicillin-binding proteins, did not appear to be essential for enzyme activity. Mutations of the Trp233 into Leu and Ser strongly decreased the enzymic activity, the affinity for beta-lactams and the protein stability. Surprisingly, the benzylpenicilloyl-(W233L)enzyme deacylated at least 300-fold more quickly than the corresponding acyl-enzyme formed with the wild-type protein and gave rise to benzylpenicilloate instead of phenylacetylglycine. This mutant DD-peptidase thus behaved as a weak beta-lactamase.

scholarly journals Revisiting the role of PML protein targeting and disruption of PML bodies in human cytomegalovirus infection

2020 ◽  
Vol 2 (7A) ◽  
Author(s):  
Christina Paulus ◽  
Thomas Harwardt ◽  
Bernadette Walter ◽  
Andrea Marxreiter ◽  
Michael Nevels
Keyword(s):  
Gene Expression ◽  
Viral Replication ◽  
Human Cytomegalovirus ◽  
Critical Role ◽  
Wild Type ◽  
Early Protein ◽  
Type Protein ◽  
Wild Type Protein ◽  
Pml Bodies

Promyelocytic leukaemia (PML) bodies are nuclear organelles implicated in post-translational modification by small ubiquitin-like modifier (SUMO) proteins and in the antiviral host cell response to infection. The 72-kDa immediate-early protein 1 (IE1) is considered the principal antagonist of PML bodies encoded by the human cytomegalovirus, one of eight human herpesviruses. Previous work has suggested that the interaction between IE1 and PML proteins, the central organisers of PML bodies, and the subsequent disruption of these organelles serve a critical role in viral replication by counteracting intrinsic antiviral immunity and the induction of interferon (IFN)-stimulated genes. However, this picture has emerged largely from studying mutant IE1 proteins known or predicted to be globally misfolded und metabolically unstable. We systematically screened for stable IE1 mutants by clustered charge-to-alanine scanning. We identified a mutant protein (IE1cc172-176) selectively defective for PML interaction. Functional comparisons between the mutant and wild-type protein revealed that IE1 can undergo modification by mixed polymeric SUMO chains and that it targets PML and Sp100, the two main constituents of PML bodies, via distinct mechanisms. Unexpectedly, IE1cc172-176 supported viral replication almost as efficiently as wild-type IE1. Moreover, lower instead of higher (as expected) levels of tumor necrosis factor alpha, IFN-beta, IFN-lambda and IFN-stimulated gene expression were observed with the mutant compared to the wild-type protein and virus. These results suggest that the disruption of PML bodies is linked to induction rather than inhibition of antiviral gene expression. Our findings challenge current views regarding the role of PML bodies in viral infection.

Conformational analysis on the wild type and mutated forms of human ORF1p: a molecular dynamics study

2015 ◽  
Vol 11 (7) ◽  
pp. 1987-1999 ◽  
Author(s):  
Rajagopalan Muthukumaran ◽  
Balasubramanian Sangeetha ◽  
Ramaswamy Amutha
Keyword(s):  
Molecular Dynamics ◽  
Complex Formation ◽  
Conformational Analysis ◽  
Structural Dynamics ◽  
Wild Type ◽  
Ribonucleoprotein Complex

Structural dynamics of human ORF1p emphasizes the role of Tyr282 in regulating ribonucleoprotein complex formation.

scholarly journals The Griffithsin Dimer Is Required for High-Potency Inhibition of HIV-1: Evidence for Manipulation of the Structure of gp120 as Part of the Griffithsin Dimer Mechanism

2013 ◽  
Vol 57 (8) ◽  
pp. 3976-3989 ◽  
Author(s):  
Jie Xue ◽  
Bart Hoorelbeke ◽  
Ioannis Kagiampakis ◽  
Borries Demeler ◽  
Jan Balzarini ◽  
...  
Keyword(s):  
Protein A ◽  
Carbohydrate Binding ◽  
Wild Type ◽  
Subtype C ◽  
Subtype B ◽  
Type Protein ◽  
Wild Type Protein ◽  
Protein Dimer ◽  
Anti Hiv

ABSTRACTGriffithsin (Grft) is a protein lectin derived from red algae that tightly binds the HIV envelope protein gp120 and effectively inhibits virus infection. This inhibition is due to the binding by Grft of high-mannose saccharides on the surface of gp120. Grft has been shown to be a tight dimer, but the role of the dimer in Grft's anti-HIV function has not been fully explored. To investigate the role of the Grft dimer in anti-HIV function, an obligate dimer of Grft was designed by expressing the protein with a peptide linker between the two subunits. This “Grft-linker-Grft” is a folded protein dimer, apparently nearly identical in structural properties to the wild-type protein. A “one-armed” obligate dimer was also designed (Grft-linker-Grft OneArm), with each of the three carbohydrate binding sites of one subunit mutated while the other subunit remained intact. While both constructed dimers retained the ability to bind gp120 and the viral surface, Grft-linker-Grft OneArm was 84- to 1,010-fold less able to inhibit HIV than wild-type Grft, while Grft-linker-Grft had near-wild-type antiviral potency. Furthermore, while the wild-type protein demonstrated the ability to alter the structure of gp120 by exposing the CD4 binding site, Grft-linker-Grft OneArm largely lost this ability. In experiments to investigate gp120 shedding, it was found that Grft has different effects on gp120 shedding for strains from subtype B and subtype C, and this might correlate with Grft function. Evidence is provided that the dimer form of Grft is critical to the function of this protein in HIV inhibition.

scholarly journals Expression and characterization of Pseudomonas aeruginosa cytochrome c-551 and two site-directed mutants: role of tryptophan 56 in the modulation of redox properties

1997 ◽  
Vol 322 (1) ◽  
pp. 35-42 ◽  
Author(s):  
Francesca CUTRUZZOLÀ ◽  
Ilaria CIABATTI ◽  
Gabriella ROLLI ◽  
Sabrina FALCINELLI ◽  
Marzia ARESE ◽  
...  
Keyword(s):  
Hydrogen Bonding ◽  
Cytochrome C ◽  
Ph Dependence ◽  
Redox Properties ◽  
Expression Vectors ◽  
Wild Type ◽  
Type Protein ◽  
Wild Type Protein ◽  
Structural And Functional Properties

The gene coding for Pseudomonas aeruginosacytochrome c-551 was expressed in Pseudomonas putidaunder aerobic conditions, using two different expression vectors; the more efficient proved to be pNM185, induced by m-toluate. Mature holo-(cytochrome c-551) was produced in high yield by this expression system, and was purified to homogeneity. Comparison of the recombinant wild-type protein with that purified from Ps. aeruginosashowed no differences in structural and functional properties. Trp56, an internal residue in cytochrome c-551, is located at hydrogen-bonding distance from haem propionate-17, together with Arg47. Ionization of propionate-17 was related to the observed pH-dependence of redox potential. The role of Trp56 in determining the redox properties of Ps. aeruginosacytochrome c-551 was assessed by site-directed mutagenesis, by substitution with Tyr (W56Y) and Phe (W56F). The W56Y mutant is similar to the wild-type cytochrome. On the other hand, the W56F mutant, although similar to the wild-type protein in spectral properties and electron donation to azurin, is characterized by a weakening of the FeŐMet61 bond, as shown in the oxidized protein by the loss of the 695 nm band approx. 2 pH units below the wild-type. Moreover, in W56F, the midpoint potential and its pH-dependence are both different from the wild-type. These results are consistent with the hypothesis that hydrogen-bonding to haem propionate-17 is important in modulation of the redox properties of Ps. aeruginosacytochrome c-551.

scholarly journals Molecular Dynamics of Chloroplast Membranes Isolated from Wild-Type Barley and a Brassinosteroid-Deficient Mutant Acclimated to Low and High Temperatures

Biomolecules ◽  
2020 ◽  
Vol 11 (1) ◽  
pp. 27
Author(s):  
Iwona Sadura ◽  
Dariusz Latowski ◽  
Jana Oklestkova ◽  
Damian Gruszka ◽  
Marek Chyc ◽  
...  
Keyword(s):  
Molecular Dynamics ◽  
Temperature Stress ◽  
High Temperatures ◽  
Biological Membrane ◽  
Hydrophobic Core ◽  
Wild Type ◽  
Chloroplast Membranes ◽  
Paramagnetic Resonance ◽  
Photosynthetic Membranes

Plants have developed various acclimation strategies in order to counteract the negative effects of abiotic stresses (including temperature stress), and biological membranes are important elements in these strategies. Brassinosteroids (BR) are plant steroid hormones that regulate plant growth and development and modulate their reaction against many environmental stresses including temperature stress, but their role in modifying the properties of the biological membrane is poorly known. In this paper, we characterise the molecular dynamics of chloroplast membranes that had been isolated from wild-type and a BR-deficient barley mutant that had been acclimated to low and high temperatures in order to enrich the knowledge about the role of BR as regulators of the dynamics of the photosynthetic membranes. The molecular dynamics of the membranes was investigated using electron paramagnetic resonance (EPR) spectroscopy in both a hydrophilic and hydrophobic area of the membranes. The content of BR was determined, and other important membrane components that affect their molecular dynamics such as chlorophylls, carotenoids and fatty acids in these membranes were also determined. The chloroplast membranes of the BR-mutant had a higher degree of rigidification than the membranes of the wild type. In the hydrophilic area, the most visible differences were observed in plants that had been grown at 20 °C, whereas in the hydrophobic core, they were visible at both 20 and 5 °C. There were no differences in the molecular dynamics of the studied membranes in the chloroplast membranes that had been isolated from plants that had been grown at 27 °C. The role of BR in regulating the molecular dynamics of the photosynthetic membranes will be discussed against the background of an analysis of the photosynthetic pigments and fatty acid composition in the chloroplasts.

scholarly journals Dynamics of the Peripheral Membrane Protein P2 from Human Myelin Measured by Neutron Scattering—A Comparison between Wild-Type Protein and a Hinge Mutant

PLoS ONE ◽  
2015 ◽  
Vol 10 (6) ◽  
pp. e0128954 ◽  
Author(s):  
Saara Laulumaa ◽  
Tuomo Nieminen ◽  
Mari Lehtimäki ◽  
Shweta Aggarwal ◽  
Mikael Simons ◽  
...  
Keyword(s):  
Membrane Protein ◽  
Neutron Scattering ◽  
Wild Type ◽  
Type Protein ◽  
Wild Type Protein ◽  
Peripheral Membrane Protein
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