The Right-Handed Parallel β-Helix Topology of Erwinia chrysanthemi Pectin Methylesterase Is Intimately Associated with Both Sequential Folding and Resistance to High Pressure.

peer reviewedThe complex topologies of large multi-domain globular proteins make the study of their folding and assembly particularly demanding. It is often characterized by complex kinetics and undesired side reactions, such as aggregation. The structural simplicity of tandem-repeat proteins, which are characterized by the repetition of a basic structural motif and are stabilized exclusively by sequentially localized contacts, has provided opportunities for dissecting their folding landscapes. In this study, we focus on the Erwinia chrysanthemi pectin methylesterase (342 residues), an all-β pectinolytic enzyme with a right-handed parallel β-helix structure. Chemicals and pressure were chosen as denaturants and a variety of optical techniques were used in conjunction with stopped-flow equipment to investigate the folding mechanism of the enzyme at 25 °C. Under equilibrium conditions, both chemical- and pressure-induced unfolding show two-state transitions, with average conformational stability (ΔG° = 35 ± 5 kJ·mol-1) but exceptionally high resistance to pressure (Pm = 800 ± 7 MPa). Stopped-flow kinetic experiments revealed a very rapid (τ < 1 ms) hydrophobic collapse accompanied by the formation of an extended secondary structure but did not reveal stable tertiary contacts. This is followed by three distinct cooperative phases and the significant population of two intermediate species. The kinetics followed by intrinsic fluorescence shows a lag phase, strongly indicating that these intermediates are productive species on a sequential folding pathway, for which we propose a plausible model. These combined data demonstrate that even a large repeat protein can fold in a highly cooperative manner

Study of the folding properties of a right-handed parallel beta helix pectin methylesterase from Erwinia chrysanthemi 3937

L’étude du repliement des protéines a pour objectif d’élucider le processus par lequel une chaîne polypeptidique adopte spontanément sa structure tridimensionnelle fonctionnelle in vivo. Cette problématique, appelée « protein folding problem », constitue l’un des enjeux majeurs de la biologie structurale moderne. En effet, une meilleure compréhension des principes fondamentaux à l’origine du repliement des protéines pourrait avoir de nombreuses applications, que ce soit dans l’approche des diverses pathologies liées au repliement incorrect des protéines, dans l’exploitation des avancées de la recherche génomique, ou encore dans la conception de protéines avec de nouvelles fonctions. La pectine méthylestérase d’Erwinia chrysanthemi 3937 (PemA) a été choisie comme protéine modèle pour réaliser des études de repliement. Cette protéine de 37 kDa est impliquée dans la dégradation de la pectine, un composant majeur de la paroi des cellules végétales. Elle possède une structure de type hélice bêta parallèle droite, qui a été proposée comme un modèle plausible pour décrire la structure de fibres amyloïdes. La caractérisation des propriétés thermodynamiques de PemA a permis de déterminer sa stabilité conformantionnelle et les études cinétiques ont permis de proposer un modèle qui décrit le chemin de repliement emprunté par la protéine lorsqu’elle acquiert sa structure native. Dans un premier temps, nous avons développé une procédure efficace afin d’obtenir la protéine d’intérêt dans des quantités suffisantes pour réaliser les différentes études biophysiques. Le gène de PemA a été cloné dans un vecteur d’expression permettant d’améliorer les rendements de production de la protéine recombinante. Le protocole de purification a également été amélioré et nous avons mis au point un test d’activité efficace permettant de vérifier la fonctionnalité de l’enzyme.La stabilité conformationnelle de PemA a été évaluée en déstabilisant la protéine à l’aide de différents agents dénaturants (chlorure de guanidine, urée, température, pH, pression), tout en mesurant l’intégrité de sa structure à l’aide d’une ou plusieurs techniques(s) spectroscopique(s) (fluorescence, dichroïsme circulaire et spectroscopie FTIR). Les résultats de ces études ont révélé que PemA est particulièrement résistante aux hautes pressions, alors que sa stabilité chimique et thermique sont dans la gamme de ce qui est généralement observé avec les protéines mésophiles. D’autre part, la caractérisation des cinétiques de repliement PemA a permis de proposer un mécanisme de repliement. La protéine se replie selon un chemin séquentiel, caractérisé par la formation d’au moins deux intermédiaires cinétiques. La simulation des cinétiques de repliement, sur la base des données expérimentales, est en bon accord avec le modèle à quatre états proposé pour décrire le repliement de PemA.The aim of protein folding studies is to elucidate the process by which a polypeptide chain spontaneously adopts its functional three-dimensional structure in vivo. This issue, known as the « protein folding problem », is one of the major challenges in modern structural biology. Indeed, a better understanding of the fundamental principles underlying protein folding could have many applications, either in the understanding of various diseases related to protein misfolding, in the exploitation of advances in genomics, or in the design of proteins with new functions. Pectin methylesterase of Erwinia chrysanthemi 3937 (PemA) was chosen as a model protein for folding studies. It is a 37 kDa protein that is involved in the degradation of pectin, a major component of the plant cell wall. This enzyme has a right-handed parallel beta-helix structure, which has been proposed as a plausible model to describe the structure of amyloid fibrils. Characterization of PemA thermodynamic properties led to the determination of its conformationnal stability, whereas kinetic studies highlighted occurence of at least two obligatory intermediates on the folding patway of the the protein when it acquires its native structure. We first developed an effective procedure to obtain the protein of interest in sufficient quantities to perform various biophysical studies. PemA gene was cloned into an expression vector allowing improved production yields of the recombinant protein. The purification protocol was also improved and we developed an efficient activity test in order to probe the functionality of the enzyme.The conformational stability of PemA was assessed by destabilizing the native protein using different denaturing agents (guanidinium chloride, urea, temperature, pH, pressure) and probing the structural integrity of the protein with the help of one or more spectroscopic technique(s) (fluorescence, circular dichroism and FTIR spectroscopy). Data showed that PemA is particularly resistant to high pressure, while its chemical and thermal stability are in the range of what is commonly observed for mesophilic proteins. On the other hand, characterization of PemA folding kinetics highlighted that it folds along a sequential pathway, characterized by the formation of at least two obligatory kinetic intermediates. Simulation of the kinetics on the basis of the experimental data reinforced the view that PemA folding is adequatly described by a four states model.Etude des propriétés de repliement d'une protéine hélice bêta parallèle droite la pectine méthylestérase d'Erwinia chrysanthemi 393

The right-handed parallel \u3b2-helix topology of Erwinia chrysanthemi pectin methylesterase Is intimately associated with both sequential folding and resistance to high pressure

The complex topologies of large multi-domain globular proteins make the study of their folding and assembly particularly demanding. It is often characterized by complex kinetics and undesired side reactions, such as aggregation. The structural simplicity of tandem-repeat proteins, which are characterized by the repetition of a basic structural motif and are stabilized exclusively by sequentially localized contacts, has provided opportunities for dissecting their folding landscapes. In this study, we focus on the Erwinia chrysanthemi pectin methylesterase (342 residues), an all-β pectinolytic enzyme with a right-handed parallel β-helix structure. Chemicals and pressure were chosen as denaturants and a variety of optical techniques were used in conjunction with stopped-flow equipment to investigate the folding mechanism of the enzyme at 25 °C. Under equilibrium conditions, both chemical- and pressure-induced unfolding show two-state transitions, with average conformational stability (ΔG° = 35 ± 5 kJ·mol−1) but exceptionally high resistance to pressure (Pm = 800 ± 7 MPa). Stopped-flow kinetic experiments revealed a very rapid (τ < 1 ms) hydrophobic collapse accompanied by the formation of an extended secondary structure but did not reveal stable tertiary contacts. This is followed by three distinct cooperative phases and the significant population of two intermediate species. The kinetics followed by intrinsic fluorescence shows a lag phase, strongly indicating that these intermediates are productive species on a sequential folding pathway, for which we propose a plausible model. These combined data demonstrate that even a large repeat protein can fold in a highly cooperative manner

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