Ion-induced conformational and stability changes in Nereis sarcoplasmic calcium binding protein: evidence that the APO state is a molten globule.
Nereis sarcoplasmic Ca(2+)-binding protein (NSCP) is a calcium buffer protein that binds Ca(2+) ions with high affinity but is also able to bind Mg(2+) ions with high positive cooperativity. We investigated the conformational and stability changes induced by the two metal ions. The thermal reversible unfolding, monitored by circular dichroism spectroscopy, shows that the thermal stability is maximum at neutral pH and increases in the order apo < Mg(2+) < Ca(2+). The stability against chemical denaturation (urea, guanidinium chloride) studied by circular dichroism or intrinsic fluorescence was found to have a similar ion dependence. To explore in more detail the structural basis of stability, we used the fluorescent probes to evaluate the hydrophobic surface exposure in the different ligation states. The apo-NSCP exhibits accessible hydrophobic surfaces, able to bind fluorescent probes, in clear contrast with denatured or Ca(2+)/Mg(2+)-bound states. Gel filtration experiments showed that, although the metal-bound NSCP has a hydrodynamic volume in agreement with the molecular mass, the volume of the apo form is considerably larger. The present results demonstrate that the apo state has many properties in common with the molten globule. The possible factors of the metal-dependent structural changes and stability are discussed.
Christova
P
P
INSERM U350 & Institut Curie-Recherche, Orsay, France.
Cox
J A
JA
Craescu
C T
CT
D000818:Animals; D000845:Annelida / Q000737:chemistry; D002118:Calcium / Q000378:metabolism; D002135:Calcium-Binding Proteins / Q000737:chemistry*; D002850:Chromatography, Gel; D002942:Circular Dichroism; D004305:Dose-Response Relationship, Drug; D005456:Fluorescent Dyes / Q000378:metabolism; D019791:Guanidine / Q000378:metabolism; D006863:Hydrogen-Ion Concentration; D007477:Ions; D009682:Magnetic Resonance Spectroscopy; D008958:Models, Molecular; D011487:Protein Conformation; D011489:Protein Denaturation; D017510:Protein Folding; D012519:Sarcoplasmic Reticulum / Q000737:chemistry*; D013050:Spectrometry, Fluorescence; D013696:Temperature; D013816:Thermodynamics; D014508:Urea / Q000378:metabolism
D002135:Calcium-Binding Proteins; D005456:Fluorescent Dyes; D007477:Ions; D014508:Urea; D019791:Guanidine; D002118:Calcium