56 research outputs found

    structural insights into n terminal to c terminal interactions and implications for thermostability of a β α 8 triosephosphate isomerase barrel enzyme

    Get PDF
    Although several factors have been suggested to contribute to thermostability, the stabilization strategies used by proteins are still enigmatic. Studies on a recombinant xylanase from Bacilllus sp. NG-27 (RBSX), which has the ubiquitous (beta/alpha)(8)-triosephosphate isomerase barrel fold, showed that just a single mutation, V1L, although not located in any secondary structural element, markedly enhanced the stability from 70 degrees C to 75 degrees C without loss of catalytic activity. Conversely, the V1A mutation at the same position decreased the stability of the enzyme from 70 degrees C to 68 degrees C. To gain structural insights into how a single extreme N-terminus mutation can markedly influence the thermostability of the enzyme, we determined the crystal structure of RBSX and the two mutants. On the basis of computational analysis of their crystal structures, including residue interaction networks, we established a link between N-terminal to C-terminal contacts and RBSX thermostability. Our study reveals that augmenting N-terminal to C-terminal noncovalent interactions is associated with enhancement of the stability of the enzyme. In addition, we discuss several lines of evidence supporting a connection between N-terminal to C-terminal noncovalent interactions and protein stability in different proteins. We propose that the strategy of mutations at the termini could be exploited with a view to modulate stability without compromising enzymatic activity, or in general, protein function in diverse folds where N and C termini are in close proximity. Database The coordinates of RBSX, V1A and V1L have been deposited in the PDB database under the accession numbers 4QCE, 4QCF, and 4QDM, respectivel

    The Critical Role of Partially Exposed N-Terminal Valine Residue in Stabilizing GH10 Xylanase from Bacillus sp.NG-27 under Poly-Extreme Conditions

    Get PDF
    BACKGROUND: Understanding the mechanisms that govern protein stability under poly-extreme conditions continues to be a major challenge. Xylanase (BSX) from Bacillus sp. NG-27, which has a TIM-barrel structure, shows optimum activity at high temperature and alkaline pH, and is resistant to denaturation by SDS and degradation by proteinase K. A comparative circular dichroism analysis was performed on native BSX and a recombinant BSX (R-BSX) with just one additional methionine resulting from the start codon. The results of this analysis revealed the role of the partially exposed N-terminus in the unfolding of BSX in response to an increase in temperature. METHODOLOGY: We investigated the poly-extremophilicity of BSX to deduce the structural features responsible for its stability under one set of conditions, in order to gain information about its stability in other extreme conditions. To systematically address the role of the partially exposed N-terminus in BSX stability, a series of mutants was generated in which the first hydrophobic residue, valine (Val1), was either deleted or substituted with various amino acids. Each mutant was subsequently analyzed for its thermal, SDS and proteinase K stability in comparison to native BSX. CONCLUSIONS: A single conversion of Val1 to glycine (Gly) changed R-BSX from being thermo- and alkali- stable and proteinase K and SDS resistant, to being thermolabile and proteinase K-, alkali- and SDS- sensitive. This result provided insight into the structure-function relationships of BSX under poly-extreme conditions. Molecular, biochemical and structural data revealed that the poly-extremophilicity of BSX is governed by a partially exposed N-terminus through hydrophobic interactions. Such hitherto unidentified N-terminal hydrophobic interactions may play a similar role in other proteins, especially those with TIM-barrel structures. The results of the present study are therefore of major significance for protein folding and protein engineering

    Plasma peptidome profiling of acute hepatitis E patients by MALDI-TOF/TOF

    Get PDF
    Background Hepatitis E is endemic to resource-poor regions, where it manifests as sporadic cases and large waterborne outbreaks. The disease severity ranges from acute self-limited hepatitis with low mortality to fulminant hepatic failure with high mortality. It is believed that the host response plays an important role in determining the progression and outcome of this disease. We profiled the plasma peptidome from hepatitis E patients to discover suitable biomarkers and understand disease pathogenesis. Results The peptidome (< 10 kDa) fraction of plasma was enriched and analyzed by mass spectrometry. A comparative analysis of the peptide pattern of hepatitis E patients versus healthy controls was performed using ClinPro Tools. We generated a peptide profile that could be used for selective identification of hepatitis E cases. We have identified five potential biomarker peaks with m/z values of 9288.6, 7763.6, 4961.5, 1060.572 and 2365.139 that can be used to reliably differentiate between hepatitis E patients and controls with areas under the receiver operating characteristic curve (AUROC) values of 1.00, 0.954, 0.989, 0.960 and 0.829 respectively. A number of proteins involved in innate immunity were identified to be differentially present in the plasma of patients compared to healthy controls. Conclusions Besides the utility of this approach for biomarker discovery, identification of changes in endogenous peptides in hepatitis E patient plasma has increased our understanding of disease pathogenesis. We have identified peptides in plasma that can reliably distinguish hepatitis E patients from healthy controls. Results from this and an earlier proteomics study are discussed

    The Critical Role of N- and C-Terminal Contact in Protein Stability and Folding of a Family 10 Xylanase under Extreme Conditions

    Get PDF
    Stabilization strategies adopted by proteins under extreme conditions are very complex and involve various kinds of interactions. Recent studies have shown that a large proportion of proteins have their N- and C-terminal elements in close contact and suggested they play a role in protein folding and stability. However, the biological significance of this contact remains elusive.In the present study, we investigate the role of N- and C-terminal residue interaction using a family 10 xylanase (BSX) with a TIM-barrel structure that shows stability under high temperature, alkali pH, and protease and SDS treatment. Based on crystal structure, an aromatic cluster was identified that involves Phe4, Trp6 and Tyr343 holding the N- and C-terminus together; this is a unique and important feature of this protein that might be crucial for folding and stability under poly-extreme conditions. folding and activity. Alanine substitution with Phe4, Trp6 and Tyr343 drastically decreased stability under all parameters studied. Importantly, substitution of Phe4 with Trp increased stability in SDS treatment. Mass spectrometry results of limited proteolysis further demonstrated that the Arg344 residue is highly susceptible to trypsin digestion in sensitive mutants such as ΔF4, W6A and Y343A, suggesting again that disruption of the Phe4-Trp6-Tyr343 (F-W-Y) cluster destabilizes the N- and C-terminal interaction. Our results underscore the importance of N- and C-terminal contact through aromatic interactions in protein folding and stability under extreme conditions, and these results may be useful to improve the stability of other proteins under suboptimal conditions

    Glyoxalase I from Brassica juncea: molecular cloning, regulation and its over-expression confer tolerance in transgenic tobacco under stress

    No full text
    Despite its ubiquitous presence, the role of glyoxalase I has not been well investigated in plants. In order to find out its physiological functions, we have cloned and characterized a cDNA from Brassica juncea encoding glyoxalase I (Gly I) and made transgenic tobacco plants harbouring Gly I in both sense and antisense orientation. The transgenic nature of the plants was confirmed by Southern blotting, and the estimated number of genes inserted ranged from one to six. The transcript and protein levels of glyoxalase I were also monitored in transgenic plants. The expression of glyoxalase I in B. juncea was upregulated in response to salt, water and heavy metal stresses. In response to a high concentration of salt, the transcript level averaged threefold higher in 72 h, and an increase in the protein was also seen by immunoblotting. The transgenic plants over-expressing glyoxalase I showed significant tolerance to methylglyoxal and high salt, as tested in detached leaf disc senescence assay. A comparison of plants expressing high and low levels of glyoxalase I showed that the tolerance to different salt concentrations was correlated with the degree of glyoxalase I expression. Our results suggest an important role of glyoxalase I in conferring tolerance to plants under stress conditions

    Spectroscopi c and surface studies of catalytically active Zn<sub>1-x</sub>Mn<sub>x</sub>Fe<sub>2</sub>O<sub>4</sub> (X= 0, 0.25, 0.50, 0.75 and 1)<i> </i>ferrospinel systems

    No full text
    1088-1091Zn-Mn ferrospinel systems have been characterized by ESR, X-ray diffraction, FTIR, BET surface area and ammonia desorption methods. It has been observed that ESR spectra of systems possessing high ' x' values are highly magnetic in nature. Distortion of the system is also observed with increasing Mn content in the samples. The g-values are in the range of 2.11410 -2.11455. XRD pattern confirms FCC lattice for the system. XRD particle size decreases with increasing Mn content in the system. Two broad bands appearing at 700 and 500 cm-1 in the FTIR spectra of spinels are described to M-O stretching frequency of tetrahedral and tetrahedral sites respectively. All catalysts show weak, medium and strong acidic sites corresponding to ammonia desorption in different temperature ranges 423-523, 523-623 and 623-723 K. The acidity and surface area increases with increasing Mn content of the system. These findings have been correlated with the catalytic activity of spinels in the alkylation of few aromatic compounds

    Transgenic tobacco expressing Entamoeba histolytica calcium binding protein exhibits enhanced growth and tolerance to salt stress

    No full text
    Calcium has been recognized as an important signal in many abiotic stresses. Our earlier work indicated the presence of homologues of a novel calcium binding protein from Entamoeba histolytica (EhCaBP) and its binding proteins with kinase activity in different plants. In this paper, we demonstrate the transfer of EhCaBP cDNA into tobacco via Agrobacterium mediated transformation. The transgenic plants expressing EhCaBP proteins were identified using western blots and immuno-precipitation of the labeled EhCaBP protein. The seeds obtained from the transgenic plants exhibited enhanced rate of germination and increased growth and had 20-37% more dry weight in 3 weeks old seedlings compared to wild type controls. The seeds of transgenic plants were also able to germinate and grow on 200 mM NaCl. The dry weight of different EhCaBP expressing plants maintained on NaCl was 55-100% more as compared to wild type plants. The increased growth and tolerance to salinity conditions were correlated with the expression of EhCaBP protein in the transgenic plants
    corecore