Characterization of the denatured states distribution of neocarzinostatin by small-angle neutron scattering and differential scanning calorimetry.
The denatured states of a small globular protein, apo-neocarzinostatin (NCS), have been characterized using several techniques. Structural properties were investigated by optical spectroscopy techniques and small-angle neutron scattering (SANS), as a function of guanidinium chloride (GdmCl) concentration. SANS experiments show that in heavy water, the protein keeps its native size at GdmCl concentrations below 2.5 M. A sharp transition occurs at about 3.6 M GdmCl, and NCS behaves like an excluded volume chain above 5 M. The same behavior is observed in deuterated buffer by fluorescence and circular dichroism measurements. For the H(2)O buffer, the transition occurs with lower concentration of denaturant, the shift being about 0.6 M. 8-Anilino-1-naphthalenesulfonate (ANS) was used as a hydrophobic fluorescent probe for studying the early stages of protein unfolding. Protein denaturation modifies the fluorescence intensity of ANS, a maximum of intensity being detected close to 2 M GdmCl in hydrogenated buffer, which shows the existence of at least one intermediate state populated at the beginning of the unfolding pathway. Differential scanning calorimetry (DSC) was used to obtain thermodynamic values for NCS denaturation. The melting curves recorded between 20 and 90 degrees C in the presence of various GdmCl concentrations (0-3 M) cannot be explained by a simple two-state model. Altogether, the data presented in this paper suggest that before unfolding the protein explores a distribution of states which is centered around compact states at denaturant concentrations below 2 M in H(2)O, and then shifts to less structured states by increasing denaturant concentrations.
Russo
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Laboratoire Léon Brillouin, CE-Saclay, 91191 Gif-sur-Yvette Cedex, France.
Durand
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Calmettes
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Desmadril
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D000817:Anilino Naphthalenesulfonates / Q000737:chemistry; D000903:Antibiotics, Antineoplastic / Q000737:chemistry; D001059:Apoproteins / Q000737:chemistry; D002152:Calorimetry, Differential Scanning / Q000379:methods; D002942:Circular Dichroism; D005456:Fluorescent Dyes; D019791:Guanidine / Q000737:chemistry; D009502:Neutrons; D011489:Protein Denaturation; D017510:Protein Folding; D012542:Scattering, Radiation; D012996:Solutions; D013050:Spectrometry, Fluorescence; D009353:Zinostatin / Q000737:chemistry*
D000817:Anilino Naphthalenesulfonates; D000903:Antibiotics, Antineoplastic; D001059:Apoproteins; D005456:Fluorescent Dyes; D012996:Solutions; C027132:1-anilino-8-naphthalenesulfonate; D009353:Zinostatin; D019791:Guanidine