Human acidic fibroblast growth factor (FGF-1) is a member of the £]-trefoil superfamily and exhibits a characteristic three-fold tertiary structure symmetry. However, evidence of this symmetry is not readily apparent at the level of the primary structure. This suggests that while selective pressures may exist to retain (or converge upon) a symmetric tertiary structure, other selective pressures have resulted in divergence of the primary structure during evolution. Using intra-chain and homologue sequence comparisons for 19 members of this family of proteins, we have designed mutants of FGF-1 that constrain a subset of core-packing residues to three-fold symmetry at the level of the primary structure. The consequences of these mutations upon structure, stability, folding and unfolding kinetics have been evaluated using a combination of x-ray crystallography, differential scanning calorimetry, isothermal equilibrium denaturation and stopped flow protein refolding/unfolding kinetics. An alternative core packing group has been introduced into FGF-1. The alternative core is very similar from the wild type (WT) core with regard to structure, stability, folding and unfolding kinetics. The remaining asymmetry within the protein core is related to asymmetry in the tertiary structure. The removal of tertiary structure asymmetry greatly increases protein stability and results in a conversion from three-state to a two-state folding pathway. The tertiary structure asymmetry is intimately linked to functional regions of the protein. Surprisingly, upon deletion of the functional insertions, the mutant protein is approximately 80 times more potent than the wild type form as determined by functional bioassays. The results show that the ƒÒ-trefoil superfold is compatible with a three-fold symmetric constraint upon the core region, as might be the case if the superfold arose as a result of gene duplication/fusion events. Furthermore, this new protein arrangement can form the basis of a structural "building block" that can greatly simplify the de novo design of ƒÒ-trefoil proteins by utilizing symmetric structural complementarity. This study implies that a symmetric architecture of the £]-trefoil fold is kinetically and thermodynamically ¡§fit¡¨. / Dissertation / PhD
| Identifer | oai:union.ndltd.org:fsu.edu/oai:fsu.digital.flvc.org:fsu_15702 |
| Creators | Brych, Stephen Robert |
| Source Sets | Florida State University |
| Detected Language | English |
| Type | Text |
| Rights | unrestricted |
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