STRUCTURE-BASED PREDICTION OF PROTEIN-FOLDING INTERMEDIATES

The complete unfolding of a protein involves the disruption of non-cov alent intramolecular interactions within the protein and the subsequen t hydration of the backbone and amino acid side-chains. The magnitude of the thermodynamic parameters associated with this process is known accurately for a growing number of globular proteins for which high-re solution structures are also available. The existence of this database of structural and thermodynamic information has facilitated the devel opment of statistical procedures aimed at quantifying the relationship s existing between protein structure and the thermodynamic parameters of folding/unfolding. Under some conditions proteins do not unfold com pletely, giving rise to states (commonly known as molten globules) in which the molecule retains some secondary structure and remains in a c ompact configuration after denaturation. This phenomenon is reflected in the thermodynamics of the process. Depending on the nature of the r esidual structure that exists after denaturation, the observed enthalp y, entropy and heat capacity changes will deviate in a particular and predictable way from the values expected for complete unfolding. For s everal proteins, these deviations have been shown to exhibit similar c haracteristics, suggesting that their equilibrium folding intermediate s exhibit some common structural features. Employing empirically deriv ed structure-energetic relationships, it is possible to identify in th e native structure of the protein those regions with the higher probab ility of being structured in equilibrium partly folded states. In this work, a thermodynamic search algorithm aimed at identifying the struc tural determinants of the molten globule state has been applied to six globular proteins; alpha-lactalbumin, barnase, IIIGlc, interleukin-1 beta, phage T4 lysozyme and phage 434 repressor. Remarkably, the struc tural features of the predicted equilibrium intermediates coincide to a large extent with the known structural features of the corresponding intermediates determined by NMR hydrogen-exchange experiments.