The objective of this research program is to seek a greater understanding of the significance of non-random structure present in the unfolded states of proteins for protein stability and folding. The FK506 binding protein (FKBP) provides a unique system to identify such correlations. High resolution structures of FKBP in both the crystal and solution forms are available and the equilibrium unfolding of FKBP has been thoroughly characterized using a variety of spectroscopic and biochemical methods. In addition, a detailed structural study of FKBP unfolded in concentrated urea and guanidine hydrochloride has been performed, demonstrating the presence of non-random structure associated with specific residues. The location of the non-random structure in unfolded FKBP was correlated with similar structures observed in the folded form, and was more strongly correlated with the propensity for helical and turn conformations predicted from statistical and thermodynamic scales of secondary structure prediction. On the other hand, a particularly surprising finding from these initial studies was that different secondary structures are formed in the folded and unfolded states for the C-terminal residues. Mutagenesis and spectroscopic approaches will be combined to further characterize the unfolded form of FKBP and to explore these relationships in detail. The previous structural and thermodynamic studies of folded and unfolded FKBP will be used to understand the role that non-random structure in the unfolded state plays in the folding and stability of FKBP. The role of non-random structures in altering native state stability in FKBP will identify general rules for similar structures in other proteins. In addition, deciphering the thermodynamic driving forces responsible for the conformational switch in the C-terminal residue upon folding will have implications in protein design and in three dimensional structure prediction.
