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.
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 A 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 A 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.
CNRS Laboratoire de Bioénergétique et Ingénierie des Protéines, UPR 9036, Institut de Microbiologie de la Méditerranée, Aix-Marseille Universités, Marseille, France.
Sebastien
S
Ranaldi
Valérie
V
Belle
Mireille
M
Woudstra
Raphael
R
Bourgeas
Bruno
B
Guigliarelli
Philippe
P
Roche
Hervé
H
Vezin
Fredéric
F
Carrière
André
A
Fournel
D020134:Catalytic Domain; D004578:Electron Spin Resonance Spectroscopy; D006801:Humans; D008049:Lipase / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D008280:Magnetics; D056004:Molecular Dynamics Simulation; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D009589:Nitrogen Oxides / Q000378:metabolism; D012996:Solutions; D013113:Spin Labels; D013696:Temperature
D009589:Nitrogen Oxides; D012996:Solutions; D013113:Spin Labels; D008049:Lipase; C518354:PNLIP protein, human; C039900:nitroxyl