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 neutro n scattering (SANS), as a function of guanidinium chloride (GdmCl) concentr ation. SANS experiments show that in heavy water, the protein keeps its nat ive 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 cir cular dichroism measurements. For the H2O buffer, the transition occurs wit h lower concentration of denaturant, the shift being about 0.6 M. 8-Anilino -1-naphthalenesulfonate (ANS) was used as a hydrophobic fluorescent probe f or studying the early stages of protein unfolding. Protein denaturation mod ifies the fluorescence intensity of ANS, a maximum of intensity being detec ted close to 2 M GdmCl in hydrogenated buffer, which shows the existence of at least one intermediate state populated at the beginning of the unfoldin g pathway. Differential scanning calorimetry (DSC) was used to obtain therm odynamic values for NCS denaturation. The melting curves recorded between 2 0 and 90 degreesC in the presence of various GdmCl concentrations (0-3 M) c annot be explained by a simple two-state model. Altogether, the data presen ted in this paper suggest that before unfolding the protein explores a dist ribution of states which is centered around compact states at denaturant co ncentrations below 2 M in H2O, and then shifts to less structured states by increasing denaturant concentrations.