Amplitude of Pancreatic Lipase Lid Opening ...
Type de document :
Article dans une revue scientifique: Article original
DOI :
URL permanente :
Titre :
Amplitude of Pancreatic Lipase Lid Opening in Solution and Identification of Spin Label Conformational Subensembles by Combining Continuous Wave and Pulsed EPR Spectroscopy and Molecular Dynamics
Auteur(s) :
Ranaldi, Sebastien [Auteur]
Bioénergétique et Ingénierie des Protéines [BIP ]
Belle, Valérie [Auteur]
Bioénergétique et Ingénierie des Protéines [BIP ]
Woudstra, Mireille [Auteur]
Bioénergétique et Ingénierie des Protéines [BIP ]
Bourgeas, Raphael [Auteur]
Interactions et Modulateurs de Réponses [IMR]
Bioénergétique et Ingénierie des Protéines [BIP ]
Guigliarelli, Bruno [Auteur]
Bioénergétique et Ingénierie des Protéines [BIP ]
Roche, Philippe [Auteur]
Interactions et Modulateurs de Réponses [IMR]
Vezin, Herve [Auteur]
Laboratoire de Chimie Organique et Macromoleculaire [UMR CNRS 8009]
Carrière, Fredéric [Auteur]
Laboratoire Enzymologie Interfaciale et de Physiologie de la Lipolyse - UPR9025 [EIPL]
Fournel, André [Auteur]
Bioénergétique et Ingénierie des Protéines [BIP ]
Bioénergétique et Ingénierie des Protéines [BIP ]
Belle, Valérie [Auteur]
Bioénergétique et Ingénierie des Protéines [BIP ]
Woudstra, Mireille [Auteur]
Bioénergétique et Ingénierie des Protéines [BIP ]
Bourgeas, Raphael [Auteur]
Interactions et Modulateurs de Réponses [IMR]
Bioénergétique et Ingénierie des Protéines [BIP ]
Guigliarelli, Bruno [Auteur]
Bioénergétique et Ingénierie des Protéines [BIP ]
Roche, Philippe [Auteur]
Interactions et Modulateurs de Réponses [IMR]
Vezin, Herve [Auteur]
Laboratoire de Chimie Organique et Macromoleculaire [UMR CNRS 8009]
Carrière, Fredéric [Auteur]
Laboratoire Enzymologie Interfaciale et de Physiologie de la Lipolyse - UPR9025 [EIPL]
Fournel, André [Auteur]
Bioénergétique et Ingénierie des Protéines [BIP ]
Titre de la revue :
Biochemistry
Nom court de la revue :
Biochemistry
Numéro :
49
Pagination :
2140-2149
Éditeur :
American Chemical Society (ACS)
Date de publication :
2010-02-18
ISSN :
0006-2960
Mot(s)-clé(s) en anglais :
Salts
Peptides and proteins
Electron paramagnetic resonance spectroscopy
Conformation
Quantum mechanics
Peptides and proteins
Electron paramagnetic resonance spectroscopy
Conformation
Quantum mechanics
Discipline(s) HAL :
Chimie/Chimie théorique et/ou physique
Résumé en anglais : [en]
The opening of the lid that controls the access to the active site of human pancreatic lipase (HPL) was measured from the magnetic interaction between two spin labels grafted on this enzyme. One spin label was introduced ...
Lire la suite >The opening of the lid that controls the access to the active site of human pancreatic lipase (HPL) was measured from the magnetic interaction between two spin labels grafted on this enzyme. One spin label was introduced at a rigid position in HPL where an accessible cysteine residue (C181) naturally occurs. A second spin label was covalently bound to the mobile lid after introducing a cysteine residue at position 249 by site-directed mutagenesis. Double electron−electron resonance (DEER) experiments allowed the estimation of a distance of 19 ± 2 Å between the spin labels when bilabeled HPL was alone in a frozen solution, i.e., with the lid in the closed conformation. A magnetic interaction was however detected by continuous wave EPR experiments, suggesting that a fraction of bilabeled HPL contained spin labels separated by a shorter distance. These results could be interpreted by the presence of two conformational subensembles for the spin label lateral chain at position 249 when the lid was closed. The existence of these conformational subensembles was revealed by molecular dynamics experiments and confirmed by the simulation of the EPR spectrum. When the lid opening was induced by the addition of bile salts and colipase, a larger distance of 43 ± 2 Å between the two spin labels was estimated from DEER experiments. The distances measured between the spin labels grafted at positions 181 and 249 were in good agreement with those estimated from the known X-ray structures of HPL in the closed and open conformations, but for the first time, the amplitude of the lid opening was measured in solution or in a frozen solution in the presence of amphiphiles.Lire moins >
Lire la suite >The opening of the lid that controls the access to the active site of human pancreatic lipase (HPL) was measured from the magnetic interaction between two spin labels grafted on this enzyme. One spin label was introduced at a rigid position in HPL where an accessible cysteine residue (C181) naturally occurs. A second spin label was covalently bound to the mobile lid after introducing a cysteine residue at position 249 by site-directed mutagenesis. Double electron−electron resonance (DEER) experiments allowed the estimation of a distance of 19 ± 2 Å between the spin labels when bilabeled HPL was alone in a frozen solution, i.e., with the lid in the closed conformation. A magnetic interaction was however detected by continuous wave EPR experiments, suggesting that a fraction of bilabeled HPL contained spin labels separated by a shorter distance. These results could be interpreted by the presence of two conformational subensembles for the spin label lateral chain at position 249 when the lid was closed. The existence of these conformational subensembles was revealed by molecular dynamics experiments and confirmed by the simulation of the EPR spectrum. When the lid opening was induced by the addition of bile salts and colipase, a larger distance of 43 ± 2 Å between the two spin labels was estimated from DEER experiments. The distances measured between the spin labels grafted at positions 181 and 249 were in good agreement with those estimated from the known X-ray structures of HPL in the closed and open conformations, but for the first time, the amplitude of the lid opening was measured in solution or in a frozen solution in the presence of amphiphiles.Lire moins >
Langue :
Anglais
Audience :
Internationale
Vulgarisation :
Non
Établissement(s) :
Université de Lille
CNRS
CNRS
Collections :
Date de dépôt :
2021-06-18T08:23:24Z
2021-10-08T11:55:11Z
2021-10-08T11:55:11Z