Type of Document	Dissertation
Author	Kim, Jaewon
URN	etd-08192004-101224
Title	Structure and Biophysical Characterization of b-Turn Regions of Human Acidic Fibroblast Growth Factor
Degree	Doctor of Philosophy
Department	Chemistry and Biochemistry, Department of
Advisory Committee	Advisor Name Title Michael Blaber Committee Chair Sanford A. Safron Committee Member Thomas C. S. Keller III Committee Member Timothy M. Logan Committee Member
Keywords	Fibroblast Human Growth Factor
Date of Defense	2003-12-13
Availability	unrestricted
Abstract The structure of human acidic fibroblast growth factor (FGF-1) is classified a - trefoil structure, one of the fundamental protein superfolds. The x-ray crystal structures of wild type and various mutants of FGF-1 have been solved in five different space groups: C2, C2221, P21 (4 molecules/asu), P21 (3 molecules/asu) and P212121. These structures reveal two characteristically different conformations for the 8/9 -hairpin comprising residue positions 90-94. This region in the wild-type FGF-1 structure (P21, four molecules/asu), a His-tagged His93Gly mutant (P21, three molecules/asu) and a His-tagged Asn106Gly mutant (P212121) adopts a 3:5 -hairpin known as a type I (1-4) G1 bulge (containing a type I turn). However, a His-tagged form of wild-type FGF-1 (C2221) and a His-tagged Leu44Phe mutant (C2) adopt a 3:3 -hairpin (containing a type I' turn) for this same region. A structural feature that distinguishes these two types of -hairpin structures is the number and location of side chain positions with eclipsed Cand main chain carbonyl oxygen groups (+60°). The effects of glycine mutations upon stability, at positions within the hairpin, have been used to identify the most likely -hairpin structure in solution. The results indicate that the 3:3 -hairpin containing a type I' turn in some FGF-1 crystal forms is adopted due to the effects of crystal packing interactions. Type I' turns in the structural data bank are quite rare, and a survey of these turns reveals that a large percentage exhibit crystal contacts within 3.0Å. The results suggest that many of the type I' turns in x-ray structures may be adopted due to crystal packing effects. However, the results also suggest that type I' tur ns may identify structurally dynamic regions within proteins. Specific residues in a polypeptide may be key contributors to the stability and foldability of the unique native structure. Identification and prediction of such residues is, therefore, an important area of investigation in solving the protein-folding problem. Atypical main chain conformations can help identify strain within a folded protein, and by inference, positions where unique amino acids may have a naturally high frequency of occurrence due to favorable contributions to stability and folding. Non-Gly residues located near the left-handed -helical region (L-) of the Ramachandran plot are a potential indicator of structural strain. Although many investigators have studied mutations at such positions, no consistent energetic or kinetic contributions to stability or folding have been elucidated. Here we report a study of the effects of Gly, Ala and Asn substitutions found within the L-region at a characteristic position in defined -hairpin turns within FGF-1, and demonstrate consistent effects upon stability and folding kinetics. The thermodynamic and kinetic data are compared to available data for similar mutations in other proteins, with excellent agreement. The results have identified that Gly at the i+3 position within a subset of -hairpin turns is a key contributor towards increasing the rate of folding to the native state of the polypeptide while leaving the rate of unfolding largely unchanged. Turn 4 and turn 8 regions are two turns related by the trefoil 3- fold axis of symmetry within FGF-1. These two turns have different turn conformations, even though there are no insertions or deletions within turn regions. By using Nuclear Magnetic Resonance (NMR) and Gly substitutions, we prove that the double conformation of turn 4 is not from the crystal-packing artifact. We also prove that the turn 4 region is a low energy conformation with little associated structural strain by using Gly screening and turn sequence swapping using the turn 8 sequence into the turn 4 sequence. We also show that the turn 8 region has a structural strain based on the significant effects upon both the protein stability and rate of folding or unfolding. By using three turn 4 mutant crystal structures and one turn 8 mutant analyzed by NMR, we show that the variable turn sequences within turn regions do no t affect the structure. Rather we find the adjacent residue to turn region 8 (Leu89) is the key residue for determining the turn structure. Previously, we demonstrated that by adding one more criterion (requiring the position to be the i+3 residue within a type I -hairpin), we could identify a consistent stability effect for Gly and Ala substitutions within the L-of the Ramachandran plot. We extended our target turn region to three additional 3:5 type I turn in FGF-1 to see the consistent results of previous chapter. We substituted each i+3 residues (all Gly) of three turn regions with Ala. The stability data of two-turn regions GlyAla substitution was consistent with what we expected (~10 kJ/mol destabilization effect). One mutant (Gly71Ala), however, showed an extra destabilization effect (~15 kJ/mol). We found this additional effect came from a bad contact of non-Gly residue with neighboring atoms. Nonetheless, we could identify a single structural property, independent of local sequence or neighboring effects, with a predictable effect upon protein stability.
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