High-pressure SANS and fluorescence unfolding study of calmodulin

• Published in: Biochimica et biophysica acta Vol. 1844; no. 9; pp. 1560 - 1568
• Main Authors: Gibrat, Gabriel, Hoa, Gaston Hui Bon, Craescu, Constantin T., Assairi, Liliane, Blouquit, Yves, Annighöfer, Burkhard, May, Roland P., Bellissent-Funel, Marie-Claire
• Format: Journal Article
• Language: English
• Published: Netherlands Elsevier B.V 01.09.2014
• Subjects: Anilino Naphthalenesulfonates; Apoproteins - chemistry; Calcium - chemistry; Calmodulin; Calmodulin - chemistry; Circular Dichroism; Conformation; Fluorescence; Fluorescent Dyes; Humans; Models, Molecular; Neutron Diffraction; Pressure; Pressure denaturation; Protein; Protein Conformation; Protein Denaturation; Protein Folding; Protein Unfolding; Recombinant Proteins - chemistry; Scattering, Small Angle; Small-angle neutron scattering; Spectrometry, Fluorescence; Temperature; Thermodynamics; Fluorescence; Pressure denaturation; Small-angle neutron scattering; Protein; Calmodulin; Conformation
• ISSN: 1570-9639; 0006-3002; 1878-1454
• DOI: 10.1016/j.bbapap.2014.05.007

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. •Pressure denaturation of calmodulin is studied by SANS, ANS and Tyr intrinsic fluorescence.•One compact intermediate state at 3000bar is found for the two calmodulin forms.•The structures under pressure of the apo- and holo-calmodulin are different.•The pressure unfolding of both calmodulin forms has been found to be reversible.