Structure Dynamics Guided Enzyme Improvement of ENDO-BETA-1, 4-XYLANASE I (original) (raw)
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HDX-Analyzer: a novel package for statistical analysis of protein structure dynamics
BMC Bioinformatics, 2011
Background: HDX mass spectrometry is a powerful platform to probe protein structure dynamics during ligand binding, protein folding, enzyme catalysis, and such. HDX mass spectrometry analysis derives the protein structure dynamics based on the mass increase of a protein of which the backbone protons exchanged with solvent deuterium. Coupled with enzyme digestion and MS/MS analysis, HDX mass spectrometry can be used to study the regional dynamics of protein based on the m/z value or percentage of deuterium incorporation for the digested peptides in the HDX experiments. Various software packages have been developed to analyze HDX mass spectrometry data. Despite the progresses, proper and explicit statistical treatment is still lacking in most of the current HDX mass spectrometry software. In order to address this issue, we have developed the HDXanalyzer for the statistical analysis of HDX mass spectrometry data using R, Python, and RPY2. Implementation and results: HDXanalyzer package contains three major modules, the data processing module, the statistical analysis module, and the user interface. RPY2 is employed to enable the connection of these three components, where the data processing module is implemented using Python and the statistical analysis module is implemented with R. RPY2 creates a low-level interface for R and allows the effective integration of statistical module for data processing. The data processing module generates the centroid for the peptides in form of m/z value, and the differences of centroids between the peptides derived from apo and ligand-bound protein allow us to evaluate whether the regions have significant changes in structure dynamics or not. Another option of the software is to calculate the deuterium incorporation rate for the comparison. The two types of statistical analyses are Paired Student's t-test and the linear combination of the intercept for multiple regression and ANCOVA model. The user interface is implemented with wxpython to facilitate the data visualization in graphs and the statistical analysis output presentation. In order to evaluate the software, a previously published xylanase HDX mass spectrometry analysis dataset is processed and presented. The results from the different statistical analysis methods are compared and shown to be similar. The statistical analysis results are overlaid with the three dimensional structure of the protein to highlight the regional structure dynamics changes in the xylanase enzyme.
Biochemistry, 2004
Structural properties and thermal stability of Trichoderma reesei endo-1,4--xylanase II (TRX II) and its three recombinant mutants were characterized using electrospray ionization Fourier transform ion cyclotron resonance (ESI FT-ICR) mass spectrometry and hydrogen/deuterium (H/D) exchange reactions. TRX II has been previously stabilized by a disulfide bridge C110-C154 and other site-directed mutations (TRX II mutants DS2 and DS5). Very recently, a highly thermostable mutant was introduced by combining mutations of DS5 with an N-terminal disulfide bridge C2-C28 (mutant DB1). Accurate mass measurements of TRX II, DS2, DS5, and DB1 verified the expected DNA-encoded protein sequences (average mass error 1.3 ppm) and allowed unequivocal assignment of the disulfides without chemical reduction and subsequent alkylation of the expected cross-links. Moreover, H/D exchange reactions provided means for the detection of a major heat-induced conformational change comprising two interconverting conformers of very different H/D exchange rates as well as allowed the apparent melting temperatures (T m ) to be determined (62.6, 65.1, 68.0, and 82.2°C for TRX II, DS2, DS5, and DB1, respectively). Residual activity measurements verified that the enzymes inactivated at significantly lower temperatures than expected on the basis of the apparent T m values, strongly suggesting that the inactivation takes place through minor conformational change other than observed by H/D exchange. ESI FT-ICR analyses also revealed molecular heterogeneity in DS5 and DB1 due to the propeptide incorporation. Resulting unintentional N-terminal extensions were observed to further improve the stability of the DB1 mutant. The extension of six amino acid residues upstream from the protein N-terminus increased stability by ∼5°C
Enzyme structure dynamics of xylanase I from Trichoderma longibrachiatum
BMC Bioinformatics, 2010
Background: Enzyme dynamics has recently been shown to be crucial for structure-function relationship. Among various structure dynamics analysis platforms, HDX (hydrogen deuterium exchange) mass spectrometry stands out as an efficient and high-throughput way to analyze protein dynamics upon ligand binding. Despite the potential, limited research has employed the HDX mass spec platform to probe regional structure dynamics of enzymes. In particular, the technique has never been used for analyzing cell wall degrading enzymes. We hereby used xylanase as a model to explore the potential of HDX mass spectrometry for studying cell wall degrading enzymes.
Mass Spectrometry, 2019
Hydrogen/deuterium exchange (HDX) coupled with pepsin digestion is useful for rapidly analyzing the kinetic properties of small amounts of protein. However, the analysis of HDX by matrix-assisted laser desorption/ionization (MALDI) mass spectrometry (MS) is time-consuming due to a lack of dedicated so ware. Currently available so ware programs mainly calculate average mass shi s, even though the isotopic distribution width contains information regarding multiple protein conformations. Moreover, HDX reaction samples are typically composed of peptides that contain various numbers of deuterium atoms, which also hinders the rapid and comprehensive analysis of protein dynamics. We report here on the development of a so ware program "Scipas DX" that can be used to automatically analyze the hydrogen-deuterium isotopic distribution in peaks in HDX spectra and calculate the average number of atoms exchanged, the average deuteration ratio, the abundance ratio for exchanged atoms, and their tted spectra with a high degree of accuracy within a few minutes. Analysis of the abundance ratio for exchanged atoms of a model protein, adenylate kinase 1, using Scipas DX indicate that the local structure at residues 83-106 and 107-117 are in a slow equilibrium, suggesting that these regions adopt multiple conformations that are involved in the stability and in switching between the active and inactive forms. Furthermore, precise HDX kinetics of the average deuteration ratio both con rmed the known induced conformations of two regions (residues 46-75 and 131-165) that are responsible for ligand binding and veri ed the novel structural dynamics of residues 107-117 and 166-196 following ligand binding to ligand-binding pockets 1 and 2, respectively. Collectively, these results highlight the usefulness and versatility of Scipas DX in MALDI-MS HDX-based analyses of protein dynamics.
Analyzing Protein Dynamics Using Hydrogen Exchange Mass Spectrometry
Journal of Visualized Experiments, 2013
All cellular processes depend on the functionality of proteins. Although the functionality of a given protein is the direct consequence of its unique amino acid sequence, it is only realized by the folding of the polypeptide chain into a single defined three-dimensional arrangement or more commonly into an ensemble of interconverting conformations. Investigating the connection between protein conformation and its function is therefore essential for a complete understanding of how proteins are able to fulfill their great variety of tasks. One possibility to study conformational changes a protein undergoes while progressing through its functional cycle is hydrogen-1 H/ 2 H-exchange in combination with high-resolution mass spectrometry (HX-MS). HX-MS is a versatile and robust method that adds a new dimension to structural information obtained by e.g. crystallography. It is used to study protein folding and unfolding, binding of small molecule ligands, protein-protein interactions, conformational changes linked to enzyme catalysis, and allostery. In addition, HX-MS is often used when the amount of protein is very limited or crystallization of the protein is not feasible. Here we provide a general protocol for studying protein dynamics with HX-MS and describe as an example how to reveal the interaction interface of two proteins in a complex.
Proteins: Structure, Function, and Genetics, 1998
Recent crystallographic studies have revealed a range of structural changes in the three-dimensional structure of endo-1,4xylanase (XYNII) from Trichoderma reesei. The observed conformational changes can be described as snapshots of an open-close movement of the active site of XYNII. These structures were further analyzed in this study. In addition, a total of four 1 ns molecular dynamics (MD) simulations were performed representing different states of the enzyme. A comparison of the global and local changes found in the X-ray structures and the MD runs suggested that the simulations reproduced a similar kind of active site opening and closing as predicted by the crystal structures. The openclose movement was characterized by the use of distance difference matrixes and the Hingefind program (Wriggers and Schulten, Proteins 29:1-14, 1997) to be a 'hinge-bending' motion involving two large rigidly-moving regions and an extended hinge. This conformational feature is probably inherent to this molecular architecture and probably plays a role in the function of XYNII. Proteins 31:434-444, 1998. 1998 Wiley-Liss, Inc.
Rapid analysis of protein structure and dynamics by hydrogen/deuterium exchange mass spectrometry
Journal of biomolecular techniques : JBT, 2003
An automated approach for the rapid analysis of protein structure has been developed and used to study acid-induced conformational changes in human growth hormone. The labeling approach involves hydrogen/deuterium exchange (H/D-Ex) of protein backbone amide hydrogens with rapid and sensitive detection by mass spectrometry (MS). Briefly, the protein is incubated for defined intervals in a deuterated environment. After rapid quenching of the exchange reaction, the partially deuterated protein is enzymatically digested and the resulting peptide fragments are analyzed by liquid chromatography mass spectrometry (LC-MS). The deuterium buildup curve measured for each fragment yields an average amide exchange rate that reflects the environment of the peptide in the intact protein. Additional analyses allow mapping of the free energy of folding on localized segments along the protein sequence affording unique dynamic and structural information. While amide H/D-Ex coupled with MS is recognize...
Journal of The American Society for Mass Spectrometry, 2012
Peptide amide hydrogen/deuterium exchange mass spectrometry (DXMS) data are often used to qualitatively support models for protein structure. We have developed and validated a method (DXCOREX) by which exchange data can be used to quantitatively assess the accuracy of three-dimensional (3-D) models of protein structure. The method utilizes the COREX algorithm to predict a protein's amide hydrogen exchange rates by reference to a hypothesized structure, and these values are used to generate a virtual data set (deuteron incorporation per peptide) that can be quantitatively compared with the deuteration level of the peptide probes measured by hydrogen exchange experimentation. The accuracy of DXCOREX was established in studies performed with 13 proteins for which both high-resolution structures and experimental data were available. The DXCOREX-calculated and experimental data for each protein was highly correlated. We then employed correlation analysis of DXCOREX-calculated versus DXMS experimental data to assess the accuracy of a recently proposed structural model for the catalytic domain of a Ca 2+-independent phospholipase A 2. The model's calculated exchange behavior was highly correlated with the experimental exchange results available for the protein, supporting the accuracy of the proposed model. This method of analysis will substantially increase the precision with which experimental hydrogen exchange data can help decipher challenging questions regarding protein structure and dynamics.
Biochemical Journal, 2001
Endo-1,4-β-xylanase II (XYNII) from Trichoderma reesei is a 21 kDa enzyme that catalyses the hydrolysis of xylan, the major plant hemicellulose. It has various applications in the paper, food and feed industries. Previous thermostability studies have revealed a significant decrease in enzymic activity of the protein at elevated temperatures in citrate buffer Enzyme Microb. Technol. 14, 566-574]. Here, thermostability of XYNII was investigated using both conventional and nanoelectrospray ionization Fourier-transform ion cyclotron resonance MS and hydrogen\deuterium (H\D)exchange reactions. In addition, dynamic light scattering (DLS) was used as a comparative method to observe possible changes in both tertiary and quaternary structures of the protein. We observed a significant irreversible conformational change and dimerization when the protein was exposed to heat. H\D exchange revealed two distinct monomeric protein populations Abbreviations used : DLS, dynamic light scattering ; ESI, electrospray ionization ; FTICR, Fourier-transform ion cyclotron resonance ; R H , hydrodynamic radius ; XYNII, endo-1,4-β-xylanase II ; H/D exchange, hydrogen/deuterium exchange.
Organic & Biomolecular Chemistry, 2009
Molecular dynamics simulations have been performed for non-covalent complexes of phenyl b-xylobioside with the retaining endo-b-1,4-xylanase from B. circulans (BCX) and its Tyr69Phe mutant using a hybrid QM/MM methodology. A trajectory initiated for the wild-type enzyme-substrate complex with the proximal xylose ring bound at the -1 subsite (adjacent to the scissile glycosidic bond) in the 4 C 1 chair conformation shows spontaneous transformation to the 2,5 B boat conformation, and potential of mean force calculations indicate that the boat is~30 kJ mol -1 lower in free energy than the chair. Analogous simulations for the mutant lacking one oxygen atom confirm the key role of Tyr69 in stabilizing the boat in preference to the 4 C 1 chair conformation, with a relative free energy difference of about 20 kJ mol -1 , by donating a hydrogen bond to the endocyclic oxygen of the proximal xylose ring. QM/MM MD simulations for phenyl b-xyloside in water, with and without a propionate/propionic acid pair to mimic the catalytic glutamate/glutamic acid pair of the enzyme, show the 4 C 1 chair to be stable, although a hydrogen bond between the OH group at C2 of xylose and the propionate moiety seems to provide some stabilization for the 2,5 B conformation. Fig. 1 Mechanism of retaining endo-1,4-b-xylanase: catalytic residues are Glu78 and Glu172. (Sugar-ring distortion not shown.) which computational modeling provides a powerful investigative tool. Many modelling studies have confirmed that substrate ring distortion is a common feature among glycosidases. 16-19 Molecular dynamics (MD) simulations have shown that the boat conformation at the -1 subsite is critical in the mechanism of family 18 chitinases, 16 and other studies have demonstrated that the -1 sugar moiety in cellulase Ce16A from Trichoderma reesi adopts a skew-boat conformation. 17 Similarly, modelling 460