Heat-induced unfolding of neocarzinostatin, a small all-beta protein investigated by small-angle X-ray scattering

Neocarzinostatin is an all-beta protein, 113 amino acid residues long, with an immunoglobulin-like fold. Its thermal unfolding has been studied by sma ll-angle X-ray scattering. Preliminary differential scanning calorimetry an d fluorescence measurements suggest that the transition is not a simple, tw o-state transition. The apparent radius of gyration is determined using thr ee different approaches, the validity of which is critically assessed using our experimental data as well as a simple, two-state model. Similarly, eac h step of data analysis is evaluated and the under lying assumptions plainl y stated. The existence of at least one intermediate state is formally demo nstrated by a singular value decomposition of the set of scattering pattern s. We assume that the pattern of the solution before the onset of the trans ition is that of the native protein, and that of the solution at the highes t temperature is that of the completely unfolded protein. Given these, actu ally not very restrictive, boundary constraints, a least-squares procedure yields a scattering pattern of the intermediate state. However, this soluti on is not unique: a whole class of possible solutions is derived by adding to the previous linear combination of the native and completely unfolded st ates. Varying the initial conditions of the least-squares calculation leads to very similar solutions. Whatever member of the class is considered, the conformation of this intermediate state appears to be weakly structured, p robably less than the transition state should be according to some proposal s. Finally, we tried and used the classical model of three thermodynamicall y well-defined states to account for our data. The failure of the simple th ermodynamic model suggests that there is more than the single intermediate structure required by singular value decomposition analysis. Formally, ther e could be several discrete intermediate species at equilibrium, or an ense mble of conformations differently populated according to the temperature. I n the latter case, a third state would be a weighted average of all non nat ive and not completely unfolded states of the protein but, since the weight s change with temperature, no meaningful curve is Likely to be derived by a global analysis using the simple model of three thermodynamically well-def ined states. (C) 2001 Academic Press.