scholarly journals The role of an evolutionarily conserved cis-proline in the thioredoxin-like domain of human class Alpha glutathione transferase A1-1

2003 ◽  
Vol 372 (1) ◽  
pp. 241-246 ◽  
Author(s):  
Chris NATHANIEL ◽  
Louise A. WALLACE ◽  
Jonathan BURKE ◽  
Heini W. DIRR
Keyword(s):  
Glutathione Transferase ◽  
Conformational Stability ◽  
Wild Type ◽  
Catalytic Function ◽  
Evolutionarily Conserved ◽  
Glutathione S Transferases ◽  
Equilibrium Unfolding ◽  
The Stability ◽  
Unfolding Kinetics

The thioredoxin-like fold has a βαβαββα topology, and most proteins/domains with this fold have a topologically conserved cis-proline residue at the N-terminus of β-strand 3. This residue plays an important role in the catalytic function and stability of thioredoxin-like proteins, but is reported not to contribute towards the stability of glutathione S-transferases (GSTs) [Allocati, Casalone, Masulli, Caccarelli, Carletti, Parker and Di Ilio (1999) FEBS Lett. 445, 347–350]. In order to further address the role of the cis-proline in the structure, function and stability of GSTs, cis-Pro-56 in human GST (hGST) A1-1 was replaced with a glycine, and the properties of the P56G mutant were compared with those of the wild-type protein. Not only was the catalytic function of the mutant dramatically reduced, so was its conformational stability, as indicated by equilibrium unfolding and unfolding kinetics experiments with urea as denaturant. These findings are discussed in the context of other thioredoxin-like proteins.

Role of the N-terminus in human 4-hydroxyphenylpyruvate dioxygenase activity

2019 ◽  
Vol 167 (3) ◽  
pp. 315-322
Author(s):  
An-Ning Feng ◽  
Chih-Wei Huang ◽  
Chi-Huei Lin ◽  
Yung-Lung Chang ◽  
Meng-Yuan Ni ◽  
...  
Keyword(s):  
Active Site ◽  
Catalytic Efficiency ◽  
Dynamics Simulation ◽  
Wild Type ◽  
Catalytic Function ◽  
C Terminus ◽  
N Terminus ◽  
Key Enzyme ◽  
The Stability

Abstract 4-Hydroxyphenylpyruvate dioxygenase (HPPD) is a key enzyme in tyrosine catabolism, catalysing the oxidation of 4-hydroxyphenylpyruvate to homogentisate. Genetic deficiency of this enzyme causes type III tyrosinaemia. The enzyme comprises two barrel-shaped domains formed by the N- and C-termini, with the active site located in the C-terminus. This study investigated the role of the N-terminus, located at the domain interface, in HPPD activity. We observed that the kcat/Km decreased ∼8-fold compared with wild type upon removal of the 12 N-terminal residues (ΔR13). Interestingly, the wild-type level of activity was retained in a mutant missing the 17 N-terminal residues, with a kcat/Km 11-fold higher than that of the ΔR13 mutant; however, the structural stability of this mutant was lower than that of wild type. A 2-fold decrease in catalytic efficiency was observed for the K10A and E12A mutants, indicating synergism between these residues in the enzyme catalytic function. A molecular dynamics simulation showed large RMS fluctuations in ΔR13 suggesting that conformational flexibility at the domain interface leads to lower activity in this mutant. These results demonstrate that the N-terminus maintains the stability of the domain interface to allow for catalysis at the active site of HPPD.

scholarly journals Equilibrium Unfolding of Neuronal Calcium Sensor-1

2005 ◽  
Vol 280 (16) ◽  
pp. 15569-15578 ◽  
Author(s):  
Dasari Muralidhar ◽  
Maroor K. Jobby ◽  
Kannan Krishnan ◽  
Vallabhaneni Annapurna ◽  
Kandala V. R. Chary ◽  
...  
Keyword(s):  
Equilibrium Model ◽  
Intracellular Signaling ◽  
Conformational Stability ◽  
Calcium Sensor ◽  
Structural Role ◽  
State Equilibrium ◽  
Neuronal Calcium Sensor ◽  
Equilibrium Unfolding ◽  
The Stability

Neuronal calcium sensor-1 (NCS-1), a Ca2+-binding protein of the calcium sensor family, modulates various functions in intracellular signaling pathways. The N-terminal glycine in this protein is myristoylated, which is presumably necessary for its physiological functions. In order to understand the structural role of myristoylation and calcium on conformational stability, we have investigated the equilibrium unfolding and refolding of myristoylated and non-myristoylated NCS-1. The unfolding of these two forms of NCS-1 in the presence of calcium is best characterized by a five-state equilibrium model, and multiple intermediates accumulate during unfolding. Calcium exerts an extrinsic stabilizing effect on both forms of the protein. In the absence of calcium, the stability of both forms is dramatically decreased, and the unfolding follows a four-state equilibrium model. The equilibrium transitions are fully reversible in the presence of calcium. Myristoylation affects the pattern of equilibrium transitions substantially but not the number of intermediates, suggesting a structural role. Our data suggest that myristoylation reduces the stiffening of the protein during initial unfolding in the presence of calcium. The effects of myristoylation are more pronounced when calcium is present, suggesting a relationship between them. Inactivating the third EF-hand motif (E120Q mutant) drastically affects the equilibrium unfolding transitions, and calcium has no effect on these transitions of the mutants. The unfolding transitions of both forms of the mutant are similar to the transitions followed by the apo forms of myristoylated and non-myristoylated NCS-1. These results suggest that the role of myristoylation in unfolding/refolding of the protein is largely dependent on the presence of calcium.

scholarly journals Role of Vif in Stability of the Human Immunodeficiency Virus Type 1 Core

2000 ◽  
Vol 74 (23) ◽  
pp. 11055-11066 ◽  
Author(s):  
Åsa Öhagen ◽  
Dana Gabuzda
Keyword(s):  
Human Immunodeficiency Virus ◽  
Dna Synthesis ◽  
Virus Entry ◽  
Wild Type ◽  
The Core ◽  
Immunodeficiency Virus ◽  
The Stability ◽  
Hiv 1

ABSTRACT The Vif protein of human immunodeficiency virus type 1 (HIV-1) is important for virion infectivity. Previous studies have shown thatvif-defective virions exhibit structural abnormalities in the virus core and are defective in the ability to complete proviral DNA synthesis in acutely infected cells. We developed novel assays to assess the relative stability of the core in HIV-1 virions. Using these assays, we examined the role of Vif in the stability of the HIV-1 core. The integrity of the core was examined following virion permeabilization or removal of the lipid envelope and treatment with various triggers, including S100 cytosol, deoxynucleoside triphosphates, detergents, NaCl, and buffers of different pH to mimic aspects of the uncoating and disassembly process which occurs after virus entry but preceding or during reverse transcription.vif mutant cores were more sensitive to disruption by all triggers tested than wild-type cores, as determined by endogenous reverse transcriptase (RT) assays, biochemical analyses, and electron microscopy. RT and the p7 nucleocapsid protein were released more readily from vif mutant virions than from wild-type virions, suggesting that the internal nucleocapsid is less stably packaged in the absence of Vif. Purified cores could be isolated from wild-type but not vif mutant virions by sedimentation through detergent-treated gradients. These results demonstrate that Vif increases the stability of virion cores. This may permit efficient viral DNA synthesis by preventing premature degradation or disassembly of viral nucleoprotein complexes during early events after virus entry.

scholarly journals Rapid capping in alpha-spectrin-deficient MEL cells from mice afflicted with hereditary hemolytic anemia.

1994 ◽  
Vol 125 (5) ◽  
pp. 1057-1065 ◽  
Author(s):  
S C Dahl ◽  
R W Geib ◽  
M T Fox ◽  
M Edidin ◽  
D Branton
Keyword(s):  
Membrane Structure ◽  
Membrane Skeleton ◽  
Wild Type ◽  
Membrane Glycoproteins ◽  
Erythroleukemia Cells ◽  
Alpha Spectrin ◽  
Erythroleukemia Cell ◽  
The Stability ◽  
In Cells

A spectrin-based membrane skeleton is important for the stability and organization of the erythrocyte. To study the role of spectrin in cells that possess complex cytoskeletons, we have generated alpha-spectrin-deficient erythroleukemia cell lines from sph/sph mice. These cells contain beta-spectrin, but lack alpha-spectrin as determined by immunoblot and Northern blot analyses. The effects of alpha-spectrin deficiency are apparent in the cells' irregular shape and fragility in culture. Capping of membrane glycoproteins by fluorescent lectin or antibodies occurs more rapidly in sph/sph than in wild-type erythroleukemia cells, and the caps appear more concentrated. The data support the idea that spectrin plays an important role in organizing membrane structure and limiting the lateral mobility of integral membrane glycoproteins in cells other than mature erythrocytes.

scholarly journals The role of tyrosine-9 and the C-terminal helix in the catalytic mechanism of Alpha-class glutathione S-transferases

1999 ◽  
Vol 343 (3) ◽  
pp. 525-531 ◽  
Author(s):  
Claire S. ALLARDYCE ◽  
Paul D. MCDONAGH ◽  
Lu-Yun LIAN ◽  
C. Roland WOLF ◽  
Gordon C. K. ROBERTS
Keyword(s):  
Conformational Change ◽  
Catalytic Mechanism ◽  
Ethacrynic Acid ◽  
Marked Decrease ◽  
Wild Type ◽  
Wild Type Enzyme ◽  
Turnover Number ◽  
Hydrophobic Substrate ◽  
Glutathione S Transferases

Glutathione S-transferases (GSTs) play a key role in the metabolism of drugs and xenobiotics. To investigate the catalytic mechanism, substrate binding and catalysis by the wild-type and two mutants of GST A1-1 have been studied. Substitution of the ‘essential’ Tyr9 by phenylalanine leads to a marked decrease in the kcat for 1-chloro-2,4-dinitrobenzene (CDNB), but has no affect on kcat for ethacrynic acid. Similarly, removal of the C-terminal helix by truncation of the enzyme at residue 209 leads to a decrease in kcat for CDNB, but an increase in kcat for ethacrynic acid. The binding of a GSH analogue increases the affinity of the wild-type enzyme for CDNB, and increases the rate of the enzyme-catalysed conjugation of this substrate with the small thiols 2-mercaptoethanol and dithiothreitol. This suggests that GSH binding produces a conformational change which is transmitted to the binding site for the hydrophobic substrate, where it alters both the affinity for the substrate and the catalytic-centre activity (‘turnover number‘) for conjugation, perhaps by increasing the proportion of the substrate bound productively. Neither of these two effects of GSH analogues are seen in the C-terminally truncated enzyme, indicating a role for the C-terminal helix in the GSH-induced conformational change.

scholarly journals Association of GSTO1 and GSTO2 Polymorphism with Risk of End-Stage Renal Disease Development and Patient Survival

2016 ◽  
Vol 35 (3) ◽  
pp. 302-311 ◽  
Author(s):  
Slavica Cimbaljevic ◽  
Sonja Suvakov ◽  
Marija Matic ◽  
Marija Pljesa-Ercegovac ◽  
Tatjana Pekmezovic ◽  
...  
Keyword(s):  
Renal Disease ◽  
End Stage Renal Disease ◽  
Cardiovascular Complications ◽  
Wild Type ◽  
Reference Allele ◽  
Kaplan Meier ◽  
Glutathione S Transferases ◽  
Stage Renal Disease ◽  
End Stage

Summary Background: Oxidative stress in patients with end-stage renal disease (ESRD) is associated with long-term cardiovascular complications. The cytosolic family of glutathione S-transferases (GSTs) is involved in the detoxication of various toxic compounds and antioxidant protection. GST omega class members, GSTO1 and GSTO2 possess, unlike other GSTs, dehydroascorbate reductase and deglutathionylation activities. The aim of this study was to clarify the role of genetic polymorphisms of GSTO1 (rs4925) and GSTO2 (rs156697) as risk determinants for ESRD development, as well as in the survival of these patients. Methods: A total of 199 patients and 199 healthy subjects were included in the study and genotyped for both GSTO1 and GSTO2 polymorphism. Protein thiol and carbonyl groups as markers of protein oxidative damage were determined spectrophotometrically. Cox proportional hazard model and Kaplan-Meier analysis were performed to investigate the role of GSTO1 and GSTO2 genetic polymorphism on mortality of ESRD patients during the follow-up period (36 month). Results: Individuals carrying the variant GSTO2 GG genotype were at 2.45-fold higher risk of ESRD development compared to the wild type GSTO2 AA genotype (OR=2.45; 95%CI=1.18-5.07; p=0.016). The results of GSTO1/GSTO2 haplotype analysis showed that the haplotype combi - nation of GSTO1 (*A)/GSTO2 (*A) (GSTO1 variant/GSTO2 wild type allele) was protective for ESRD (OR=0.23 95%CI=0.12-0.44, p=0.001). Patients carrying at least one GSTO1 reference allele have shorter mean overall (Log rank=2.844, p =0.241) and cardiovascular survival probability (Log rank=4.211, p=0.122). Conclusions: GSTO polymorphisms have been shown to act as significant markers in assessing the risk of ESRD development and patients’ survival.

scholarly journals Kinetic Studies on CphA Mutants Reveal the Role of the P158-P172 Loop in Activity versus Carbapenems

2016 ◽  
Vol 60 (5) ◽  
pp. 3123-3126 ◽  
Author(s):  
Carlo Bottoni ◽  
Mariagrazia Perilli ◽  
Francesca Marcoccia ◽  
Alessandra Piccirilli ◽  
Cristina Pellegrini ◽  
...  
Keyword(s):  
Catalytic Activity ◽  
Catalytic Efficiency ◽  
Kinetic Studies ◽  
Zinc Ion ◽  
Site Directed Mutagenesis ◽  
Directed Mutagenesis ◽  
Wild Type ◽  
The Stability

ABSTRACTSite-directed mutagenesis of CphA indicated that prolines in the P158-P172 loop are essential for the stability and the catalytic activity of subclass B2 metallo-β-lactamases against carbapenems. The sequential substitution of proline led to a decrease of the catalytic efficiency of the variant compared to the wild-type (WT) enzyme but also to a higher affinity for the binding of the second zinc ion.

scholarly journals Role of Phage Capsid in the Resistance to UV-C Radiations

2021 ◽  
Vol 22 (7) ◽  
pp. 3408
Author(s):  
Laura Maria De Plano ◽  
Domenico Franco ◽  
Maria Giovanna Rizzo ◽  
Vincenzo Zammuto ◽  
Concetta Gugliandolo ◽  
...  
Keyword(s):  
Molecular Mechanisms ◽  
3D Modelling ◽  
Wild Type ◽  
Peptide Bonds ◽  
Environmental Resistance ◽  
Modelling Analysis ◽  
Phage Capsid ◽  
The Stability ◽  
Uv C

The conformational variation of the viral capsid structure plays an essential role both for the environmental resistance and acid nuclear release during cellular infection. The aim of this study was to evaluate how capsid rearrangement in engineered phages of M13 protects viral DNA and peptide bonds from damage induced by UV-C radiation. From in silico 3D modelling analysis, two M13 engineered phage clones, namely P9b and 12III1, were chosen for (i) chemical features of amino acids sequences, (ii) rearrangements in the secondary structure of their pVIII proteins and (iii) in turn the interactions involved in phage capsid. Then, their resistance to UV-C radiation and hydrogen peroxide (H2O2) was compared to M13 wild-type vector (pC89) without peptide insert. Results showed that both the phage clones acquired an advantage against direct radiation damage, due to a reorganization of interactions in the capsid for an increase of H-bond and steric interactions. However, only P9b had an increase in resistance against H2O2. These results could help to understand the molecular mechanisms involved in the stability of new virus variants, also providing quick and necessary information to develop effective protocols in the virus inactivation for human activities, such as safety foods and animal-derived materials.

scholarly journals The Role of COA6 in the Mitochondrial Copper Delivery Pathway to Cytochrome c Oxidase

Biomolecules ◽  
2022 ◽  
Vol 12 (1) ◽  
pp. 125
Author(s):  
Abhinav B. Swaminathan ◽  
Vishal M. Gohil
Keyword(s):  
Cytochrome C ◽  
Cytochrome C Oxidase ◽  
Mitochondrial Respiratory Chain ◽  
Functional Studies ◽  
Evolutionarily Conserved ◽  
Human Disorders ◽  
The Stability ◽  
Copper Delivery

Copper is essential for the stability and activity of cytochrome c oxidase (CcO), the terminal enzyme of the mitochondrial respiratory chain. Copper is bound to COX1 and COX2, two core subunits of CcO, forming the CuB and CuA sites, respectively. Biogenesis of these two copper sites of CcO occurs separately and requires a number of evolutionarily conserved proteins that form the mitochondrial copper delivery pathway. Pathogenic mutations in some of the proteins of the copper delivery pathway, such as SCO1, SCO2, and COA6, have been shown to cause fatal infantile human disorders, highlighting the biomedical significance of understanding copper delivery mechanisms to CcO. While two decades of studies have provided a clearer picture regarding the biochemical roles of SCO1 and SCO2 proteins, some discrepancy exists regarding the function of COA6, the new member of this pathway. Initial genetic and biochemical studies have linked COA6 with copper delivery to COX2 and follow-up structural and functional studies have shown that it is specifically required for the biogenesis of the CuA site by acting as a disulfide reductase of SCO and COX2 proteins. Its role as a copper metallochaperone has also been proposed. Here, we critically review the recent literature regarding the molecular function of COA6 in CuA biogenesis.

scholarly journals Chromosome-Based Genetic Complementation System for Xylella fastidiosa

2009 ◽  
Vol 75 (6) ◽  
pp. 1679-1687 ◽  
Author(s):  
Ayumi Matsumoto ◽  
Glenn M. Young ◽  
Michele M. Igo
Keyword(s):  
Dna Sequences ◽  
Xylella Fastidiosa ◽  
Cell Physiology ◽  
Multiple Cloning Site ◽  
Wild Type ◽  
In Planta ◽  
Catalase Peroxidase ◽  
The Stability

ABSTRACT Xylella fastidiosa is a xylem-limited, gram-negative bacterium that causes Pierce's disease of grapevine. Here, we describe the construction of four vectors that facilitate the insertion of genes into a neutral site (NS1) in the X. fastidiosa chromosome. These vectors carry a colE1-like (pMB1) replicon and DNA sequences from NS1 flanking a multiple-cloning site and a resistance marker for one of the following antibiotics: chloramphenicol, erythromycin, gentamicin, or kanamycin. In X. fastidiosa, vectors with colE1-like (pMB1) replicons have been found to result primarily in the recovery of double recombinants rather than single recombinants. Thus, the ease of obtaining double recombinants and the stability of the resulting insertions at NS1 in the absence of selective pressure are the major advantages of this system. Based on in vitro and in planta characterizations, strains carrying insertions within NS1 are indistinguishable from wild-type X. fastidiosa in terms of growth rate, biofilm formation, and pathogenicity. To illustrate the usefulness of this system for complementation analysis, we constructed a strain carrying a mutation in the X. fastidiosa cpeB gene, which is predicted to encode a catalase/peroxidase, and showed that the sensitivity of this mutant to hydrogen peroxide could be overcome by the introduction of a wild-type copy of cpeB at NS1. Thus, this chromosome-based complementation system provides a valuable genetic tool for investigating the role of specific genes in X. fastidiosa cell physiology and virulence.

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