title: High-pressure SANS and fluorescence unfolding study of calmodulin.
abstract: Apo-calmodulin, a small soluble mainly α protein, is a calcium-dependent protein activator. Calcium binding affects the calmodulin conformation but also its stability. Calcium free form unfolds between 40 and 80°C, whereas the calcium-saturated form is stable up to temperatures as high as 100°C, forbidding comparison of the thermal unfolding pathways of the two forms. Thus, this paper focuses especially on the conformation of pressure-induced unfolding states of both forms of calmodulin, by combining small-angle neutron scattering (SANS) with biophysical techniques such as tyrosines and ANS fluorescence. In contrast to heat denaturation (Gibrat et al., BBA, 2012), the pressure denaturation of calmodulin is reversible up to pressures of 3000bar (300MPa). A pressure-induced compact intermediate state has been found for the two calmodulin forms, but their unfolding pathways are different. A domain compaction and an increase of the ANS fluorescence of holo form have been evidenced. On the contrary, a domain dilatation and an ANS fluorescence decrease have been found for the apo form. The pressure induced an increase of the interdomain distance for both calmodulin forms, suggesting that the central linker of calmodulin is flexible in solution.
authors.affiliation: Laboratoire Léon Brillouin, CEA/CNRS, Saclay, France.
authors.forename: Gabriel
authors.initials: G
authors.lastname: Gibrat
authors.affiliation: Inserm U779, Le Kremlin-Bicêtre, France.
authors.forename: Gaston Hui Bon
authors.initials: GH
authors.lastname: Hoa
authors.affiliation: Inserm U759/Institut Curie-Recherche, Orsay, France.
authors.forename: Constantin T
authors.initials: CT
authors.lastname: Craescu
authors.affiliation: Inserm U759/Institut Curie-Recherche, Orsay, France.
authors.forename: Liliane
authors.initials: L
authors.lastname: Assairi
authors.affiliation: Inserm U759/Institut Curie-Recherche, Orsay, France.
authors.forename: Yves
authors.initials: Y
authors.lastname: Blouquit
authors.affiliation: Laboratoire Léon Brillouin, CEA/CNRS, Saclay, France.
authors.forename: Burkhard
authors.initials: B
authors.lastname: Annighöfer
authors.affiliation: Institut Laue Langevin, Grenoble, France.
authors.forename: Roland P
authors.initials: RP
authors.lastname: May
authors.affiliation: Laboratoire Léon Brillouin, CEA/CNRS, Saclay, France.
authors.forename: Marie-Claire
authors.initials: MC
authors.lastname: Bellissent-Funel
mesh_terms: D000817:Anilino Naphthalenesulfonates; D001059:Apoproteins / Q000737:chemistry*; D002118:Calcium / Q000737:chemistry*; D002147:Calmodulin / Q000737:chemistry*; D002942:Circular Dichroism; D005456:Fluorescent Dyes; D006801:Humans; D008958:Models, Molecular; D033363:Neutron Diffraction; D011312:Pressure; D011487:Protein Conformation; D011489:Protein Denaturation; D017510:Protein Folding; D058767:Protein Unfolding; D011994:Recombinant Proteins / Q000737:chemistry; D053838:Scattering, Small Angle; D013050:Spectrometry, Fluorescence; D013696:Temperature; D013816:Thermodynamics
chemical_list: D000817:Anilino Naphthalenesulfonates; D001059:Apoproteins; D002147:Calmodulin; D005456:Fluorescent Dyes; D011994:Recombinant Proteins; C027132:1-anilino-8-naphthalenesulfonate; D002118:Calcium
keywords: Calmodulin; Conformation; Fluorescence; Pressure denaturation; Protein; Small-angle neutron scattering