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Topology, frustration, folding and function of the inflammatory cytokine Interleukin-1[beta]


One of the most intriguing problems in biology today continues to be centered on understanding the mechanism by which a newly synthesized linear polypeptide chain attains its functional three-dimensional native structure. Initially, it was thought that the fundamental problem arose because even a small protein has access to an enormous number of possible conformational states, which could not be searched through in a random way in any reasonable way in any reasonable length of time. This problem became known as the "Levinthal Paradox" because proteins do fold in a timely fashion and people are interested in the mechanism by which the primary structure directs folding of the protein to the fully folded state. In real proteins, the interplay of chain connectivity with desired structure and function creates ruggedness on the folding landscape, making a rough or partially frustrated funnel. Interleukin-1[beta] (IL-1[beta]) is an ideal system for experimental determination of the role of topology on folding mechanisms. IL-1[beta] adopts a [beta] -trefoil fold, where the tertiary fold is formed from six two-stranded hairpins, three of which form a barrel structure and the other three form a triangular array to "cap" one end of the barrel. The study of circular permutated proteins presents a particularly interesting way of testing this topology-based model. By changing the connectivity through the construction IL-1[beta] circular permutations, we studied the balance between topology and energetics on the formation of the intermediate and the overall folding mechanism. In this study, the role of topology and the affects circular permutations have on folding and the native state of IL-1[beta] was investigated. Using a combination of techniques, such as H /D, \¹\³C chemical shift analysis (CSI), stopped-flow and manual mixing fluorescence, we characterize the stability, the native state, and the folding mechanisms for several permutations. We also test the affects of the frustration of the [beta]-bulge on the native state by engineering a loop swap from IL-1ra. These studies reveal topological similarities among the variants of engineered proteins

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