臺灣博碩士論文加值系統

The three dimensional solution structure of an acidic fibroblast growth factor (nFGF-1) from the newt (Notopthalmus Viridescens) is determined using multidimensional NMR techniques. Complete assignment of the all atoms (1H, 15N and 13C) has been achieved using a variety of triple resonance experiments. A total of 50 structures were calculated by means of hybrid distance geometry-dynamical simulated annealing using a total of 1296 constraints. The atomic root mean square distribution for the 12 structures is 0.65 Å for the backbone atoms in the structured region. The secondary structural elements include 12 b-strands arranged antiparallely into a b-barrel structure. The ligand, sucrose octasulfate (SOS), binds to the protein in a stoichiometric ratio of 1:1. The protein is found to exist in a monomeric state upon binding to SOS. The SOS binding site consists of a dense cluster of positively charged residues located at the C-terminal end of the molecule. Urea-induced unfolding experiments reveal that human acidic fibroblast growth factor (hFGF-1) is structurally more stable than that of nFGF-1. The differential stability of nFGF-1 and hFGF-1 is attributed to the differences in the number of hydrogen bonds and the solvent-exposed hydrophobic residues. The antigenic diversity observed in nFGF-1 and hFGF-1 is proposed to arise from the difference(s) in the solvent accessible hydrophobic surfaces in the two homologues fibroblast growth factors (nFGF-1 and hFGF-1).
We report fibril formation in an all b-barrel protein, the newt acidic fibroblast growth factor (nFGF-1). Fibril formation is observed to be maximum in 15% (v/v) 2,2,2-trifluoroethanol (TFE). The fibrils formed by nFGF-1 are positively stained by Congo red and Thioflavine T. Electron microscopy investigation of the ultra structure of the fibrils reveals that they are about 15 nm in diameter and appears to originate from amorphous aggregates. These fibrils are found to be resistant to restricted proteolytic digestion. Results of the IR spectroscopy and far UV-CD experiments show that the fibrils possess extensive b-sheet structure. Results of the various biophysical experiments including multidimensional NMR reveal that the protein (nFGF-1) accumulates in a partially structured intermediate state(s) prior to fibril formation. The fibrils formed have very similar characteristic of amyloid fibrils and is observed to be triggered by the disruption of the native b-barrel architecture leading to the formation ‘sticky’ linear array of b-strands. We also studied the ability of anions of Hoffmeister series in prevention of aggregation and fibril formation. The inhibitory effects of these anions are studied in terms of its ability to decrease the rate of aggregation. It is found that sulfate (SO42-) ions could play a potential role in preventing or inhibiting aggregation and fibril formation. Sulfate ions prevents aggregation and fibril formation by stabilizing the native conformation of the nFGF-1.
In the present study, for the first time we demonstrate the thermal induced fibril formation in a b-barrel protein, such as the acidic fibroblast growth factor from Notopthalmus viridescens (nFGF-1). Congo red and Thioflavin T binding assays show that fibril formation occurs maximally at 65 °C. Electron microscope analysis of the thermal induced fibrils of nFGF-1 show that they have a beaded appearance. Again the thermal induced fibrils have amyloid like characters. We demonstrate that fibril formation in nFGF-1 involves the formation of a partially structured intermediate in the thermal unfolding pathway. ANS binding experiments show that the thermal induced partially structured possess solvent exposed hydrophobic surfaces. Fibril formation is proposed to occur due to the coalescence of the protein through the solvent exposed non-polar surface(s). We also, examined the efficiency of organic osmolytes in prevention of thermal induced fibril formation. Proline is found to influence the co-operativity of the thermal induced unfolding and hence decrease the population of ‘sticky’ thermal equilibrium intermediate responsible for the fibril formation process.
Refolding of the acidic fibroblast growth factor from its urea denatured state is shown to proceed via the formation of a ‘unproductive’, intermediate(s). This ‘unproductive’ intermediate cannot go back to native state upon refolding. The formation of the ‘unproductive’ intermediate is found to be independent of the nature of the denaturant and concentration of the refolding protein. 1H-15N HSQC spectra of the protein shows that the ‘unproductive’ intermediate has structural features resembling that of the denatured state. It is proposed that the ‘unproductive’ intermediate arises due to the presence of cis proline in the unfolded state(s) of the protein. Here, we used cis-trans proline isomarase to refold the protein back to its native state. We also characterized the ‘unproductive’ intermediate using variety of biophysical techniques.

Contents
1Introduction1
1.1Colloid Theory and Protein aggregation2
1.2Aggregation kinetics 4
1.3Models explaining in vitro/in vivo
protein aggregation6
1.4Protein aggregates formed in vivo8
1.5Amyloid formation9
1.6Amyloid Fibril formation requires nucleation10
1.7Firbillization occurs via Prefibrillar
intermediates11
1.8Involvement of intermediates in
Fibril formation12
1.9Models for amyloid fibril formation14
1.10Fibroblast Growth Factors15
1.11An overview of the present study21
2Materials and Methods24
2.1Expression, Purification and
Characterization of newt FGF-125
2.2Preparation of Isotope enriched newt FGF-127
2.3NMR expriments27
2.4Structure calculation29
2.5Fluorescence experiments31
2.5.1Thioflavin T (ThT) binding assay32
2.5.2ANS binding experiments32
2.5.3Urea denaturation33
2.6Circular Dichroism experiments33
2.7Equilibrium unfolding and data analysis33
2.8UV - experiments 34
2.8.1Turbidity measurements 34
2.8.2Congo red assay35
2.9Sedimentation velocity experiments35
2.10Confocal Microscopy36
2.11Scanning and Transmission electron
microscopy36
2.12Fourier Transform Infrared spectroscopy
(FT-IR)37
2.13Limited trypsin digestion38
2.14X-ray Diffraction study38
3Results and Discussion39
3.1Structure and Stability of Newt
(Notophthalmus viridescens)Acidic Fibroblast
Growth Factor40
3.1.1Molecular state of newt FGF-143
3.1.2Chemical shift assignments of newt FGF-145
3.1.3Quality of the newt FGF-1 NMR structure47
3.1.4Description of the structure51
3.1.5The newt FGF-1/SOS interaction site56
3.1.6Comparison of the structural stabilities
of newt FGF-1and human FGF-162
3.1.7Structural basis for the observed
differences in the conformational stability65
3.2Alcohol induced Aggregation
and fibril formation in newt FGF-170
3.2.1Aggregates have amyloid-like
characteristics72
3.2.2Ultrastructure of fibrils75
3.2.3Conformational transitions induced by TFE
in newt FGF-178
3.2.4Partially folded intermediates accumulate
prior to fibril formation81
3.2.5Structural features of the intermediate(s)85
3.2.6Conformational flexibility of the
intermediate(s)88
3.2.7Molecular state of the intermediate(s)91
3.2.8Structural events leading to firbril
formation93
3.2.9The effect of anions of Hoffmeister series
in the preventionof aggregation of newt FGF-199
3.3Temperature induced fibril formation
in newt FGF-1107
3.3.1Temperature induced aggregation107
3.3.2Identification of the amyloid-like fibrils108
3.3.3Ultrastructure of the thermal-induced
fibrils112
3.3.4Influence of rate of heating on fibril
formation115
3.3.5Thermal unfolding of newt FGF-1116
3.3.6Formation of partially structured state(s)119
3.3.7Structural interactions in the thermal
induced fibrils 122
3.3.8Model for the thermal induced fibril
formation in newt FGF-1124
3.3.9Effect of proline on the thermal induced
fibril formation125
3.3.10Proline affects the co-operativity of the
unfolding process129
3.4Characterization of an Intermediate in
the refolding pathway of newt FGF-1 131
4References154
5Conclusion176

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