A program is proposed that focuses on the folding mechanism of an all beta sheet protein, interleukin 1B, with the goal of elucidating the factors that direct the protein along the productive (to native state) and non-productive (to inclusion body) folding pathways. Interleukin 1B is a 153 amino acid protein having a single tryptophan residue, an assigned 1-H NMR spectrum, a solution and crystal structure, and many prepared mutants. Preliminary guanidine induced unfolding studies show an equilibrium unfolding intermediate. The investigator proposes to determine the solution structure and dynamics of this intermediate using heteronuclear NMR, time-resolved fluorescence, and solute quenching studies. Stopped flow and rapid quench kinetic methods will be used to refine the folding mechanism and to determine if an intermediate is also determined on the kinetic pathway. The population of the putative kinetic intermediate will be characterized by stopped-flow optical techniques. Following the establishment of a working kinetic model, mutation studies will be initiated to test the model and the structure of the intermediates by replacement of specific amino acid side chains to test the importance of hydrophobic interactions, packing of the beta-barrel, and ordering of surface loops in directing the pathway and maintaining the native fold. Mutant proteins will be subjected to a full equilibrium and stopped-flow analysis, and the effect of the mutation on the formation of specific intermediates in both the unfolding and refolding pathways will be determined. Since off- pathway aggregation may be correlated with the lifetimes and/or solubilities of transient intermediates in the folding pathway, the tendency to form inclusion bodies will be studied by kinetic analysis. Initial studies will focus on the Lys-97-->Val variant, which is deposited into inclusion bodies in E. coli at high levels. This mutant demonstrates increased thermodynamic stability, compared to the wild type, and identical kinetics for the rate determining last step in folding. It is proposed to study the effect of this mutation on the structure, rate of formation and solubility of the early folding intermediates by a combination of rapid-quench, stopped- flow fluorescence, and dye-binding experiments.
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