A circular dichroism study of preferential hydration and alcohol effects on a denatured protein, pig calpastatin domain I.
Pig calpastatin domain I (CSD1), a proteinase inhibitor that specifically blocks activity of calpain I and II, is a good candidate protein for studying conformational variations in the denatured form of protein. An extensive structural characterization of CSD1 reported in the first part of this work has shown that CSD1 at neutral pH is in an expanded and flexible conformation without secondary and tertiary structures. Next, we further studied cosolvent effects of protein-stabilizers, polyols and sulfate salts, as well as protein-destabilizers, alcohols, on the conformation of CSD1 monitored by far- and near-UV CD spectroscopy. We found that both groups of cosolvents at high concentration induce highly helical structures of CSD1, but without specific tertiary interactions. Based on the results on the polyols and the sulfate salts, we have suggested that the preferential hydration is one of the thermodynamic forces to induce secondary structures in the denatured state of protein. Variations in isodichroic points of changes in far-UV CD spectrum as functions of cosolvent species and their concentration have exhibited complexity of the processes. The present study implies that protein stability in the presence of cosolvents is often determined by free energy difference between the folded and the highly structured denatured state, not between the folded and the random state.
Konno
T
T
National Institute for Physiological Science, Myodaiji, Japan. konno@nips.ac.jp
Tanaka
N
N
Kataoka
M
M
Takano
E
E
Maki
M
M
D000818:Animals; D001059:Apoproteins / Q000737:chemistry; D002135:Calcium-Binding Proteins / Q000737:chemistry*; D002942:Circular Dichroism; D003574:Cytochrome c Group / Q000737:chemistry; D005990:Glycerol; D006863:Hydrogen-Ion Concentration; D000432:Methanol; D009211:Myoglobin / Q000737:chemistry; D019906:Nuclear Magnetic Resonance, Biomolecular; D011487:Protein Conformation; D011489:Protein Denaturation; D012997:Solvents; D013012:Sorbitol; D013552:Swine; D014270:Trifluoroethanol; D014961:X-Ray Diffraction
D001059:Apoproteins; D002135:Calcium-Binding Proteins; D003574:Cytochrome c Group; D009211:Myoglobin; D012997:Solvents; C010857:apomyoglobin; D013012:Sorbitol; D014270:Trifluoroethanol; C033668:calpastatin; D005990:Glycerol; D000432:Methanol