Biomedical subjects

Chin Yu

Publications and source records attributed to Chin Yu.

28 records · Page 2Linked to original sources

PubMed2003

Amyloid-like fibril formation in an all beta-barrel protein. Partially structured intermediate state(s) is a precursor for fibril formation.

Acidic fibroblast growth factor from newt (Notopthalmus viridescens) is a approximately 15-kDa, all beta-sheet protein devoid of disulfide bonds. In the present study, we investigate the effects of 2,2,2-trifluoroethanol (TFE) on the structure of newt acidic fibroblast growth factor (nFGF-1). The protein aggregates maximally in 10% (v/v) TFE. Congo red and thioflavin T binding experiments suggest that the aggregates induced by TFE have properties resembling the amyloid fibrils. Transmission electron microscopy and x-ray fiber diffraction data show that the fibrils (induced by TFE) are straight, unbranched, and have a cross-beta structure with an average diameter of 10-15 A. Preformed fibrils (induced by TFE) of nFGF-1 are observed to seed amyloid-like fibril formation in solutions containing the protein (nFGF-1) in the native beta-barrel conformation. Fluorescence, far-UV CD, anilino-8-napthalene sulfonate binding, multidimensional NMR, and Fourier transformed infrared spectroscopy data reveal that formation of a partially structured intermediate state(s) precedes the onset of the fibrillation process. The native beta-barrel structure of nFGF-1 appears to be disrupted in the partially structured intermediate state(s). The protein in the partially structured intermediate state(s) is found to be "sticky" with a solvent-exposed non-polar surface(s). Amyloid fibril formation appears to occur due to coalescence of the protein in the partially structured intermediate state(s) through solvent-exposed non-polar surfaces and intermolecular beta-sheet formation among the extended, linear beta-strands in the protein.

Amyloid beta-Peptides↗

PubMed2003

Structurally homologous all beta-barrel proteins adopt different mechanisms of folding.

Acidic fibroblast growth factors from human (hFGF-1) and newt (nFGF-1) (Notopthalamus viridescens) are 16-kDa, all beta-sheet proteins with nearly identical three-dimensional structures. Guanidine hydrochloride (GdnHCl)-induced unfolding of hFGF-1 and nFGF-1 monitored by fluorescence and far-UV circular dichroism (CD) shows that the FGF-1 isoforms differ significantly in their thermodynamic stabilities. GdnHCl-induced unfolding of nFGF-1 follows a two-state (Native state to Denatured state(s)) mechanism without detectable intermediate(s). By contrast, unfolding of hFGF-1 monitored by fluorescence, far-UV circular dichroism, size-exclusion chromatography, and NMR spectroscopy shows that the unfolding process is noncooperative and proceeds with the accumulation of stable intermediate(s) at 0.96 M GdnHCl. The intermediate (in hFGF-1) populated maximally at 0.96 M GdnHCl has molten globule-like properties and shows strong binding affinity to the hydrophobic dye, 1-Anilino-8-naphthalene sulfonate (ANS). Refolding kinetics of hFGF-1 and nFGF-1 monitored by stopped-flow fluorescence reveal that hFGF-1 and nFGF-1 adopts different folding mechanisms. The observed differences in the folding/unfolding mechanisms of nFGF-1 and hFGF-1 are proposed to be either due to differential stabilizing effects of the charged denaturant (Gdn(+) Cl(-)) on the intermediate state(s) and/or due to differences in the structural interactions stabilizing the native conformation(s) of the FGF-1 isoforms.

Amino Acid Sequence↗

PubMed2002

Investigation of the structural stability of the human acidic fibroblast growth factor by hydrogen-deuterium exchange.

The conformational stability of the human acidic fibroblast growth factor (hFGF-1) is investigated using amide proton exchange and temperature-dependent chemical shifts, monitored by two-dimensional NMR spectroscopy. The change in free energy of unfolding (DeltaG(u)) of hFGF-1 is estimated to be 5.00 +/- 0.09 kcal.mol(-)(1). Amide proton-exchange rates of 74 residues (in hFGF-1) have been unambiguously measured, and the exchange process occurs predominately according to the conditions of the EX2 limit. The exchange rates of the fast-exchanging amide protons exposed to the solvent have been measured using the clean SEA-HSQC technique. The amide proton protection factor and temperature coefficient estimates show reasonably good correlation. Residues in beta-strands II and VI appear to constitute the stability core of the protein. Among the 12 beta-strands constituting the beta-barrel architecture of hFGF-1, beta-strand XI, located in the heparin binding domain, exhibits the lowest average protection factor value. Amide protons involved in the putative folding nucleation site in hFGF-1, identified by quench-flow NMR studies, do not represent the slow-exchanging core. Residues in portions of hFGF-1 experiencing high conformational flexibility mostly correspond to those involved in receptor recognition and binding.

Amides↗

PubMed2002

Characterization of the structure and dynamics of a near-native equilibrium intermediate in the unfolding pathway of an all beta-barrel protein.

The structure and dynamics of equilibrium intermediate in the unfolding pathway of the human acidic fibroblast growth factor (hFGF-1) are investigated using a variety of biophysical techniques including multidimensional NMR spectroscopy. Guanidinium hydrochloride (GdnHCl)-induced unfolding of hFGF-1 proceeds with the accumulation of a stable intermediate state. The transition from the intermediate state to the unfolded state(s) is cooperative without the accumulation of additional intermediate(s). The intermediate state induced maximally in 0.96 m GdnHCl is found to be obligatory in the folding/unfolding pathway of hFGF-1. Most of the native tertiary structure interactions are preserved in the intermediate state. (1)H-(15)N chemical shift perturbation data suggest that the residues in the C-terminal segment including those located in the beta-strands IX, X, and XI undergo the most discernible structural change(s) in the intermediate state in 0.96 m GdnHCl. hFGF-1 in the intermediate state (0.96 m GdnHCl) does not bind to its ligand, sucrose octasulfate. Limited proteolytic digestion experiments and hydrogen-deuterium exchange monitored by (15)N heteronuclear single quantum coherence (HSQC) spectra show that the conformational flexibility of the protein in the intermediate state is significantly higher than in the native conformation. (15)N spin relaxation experiments show that many residues located in beta-strands IX, X, and XI exhibit conformational motions in the micro- to millisecond time scale. Analysis of (15)N relaxation data in conjunction with the amide proton exchange kinetics suggests that residues in the beta-strands II, VIII, and XII possibly constitute the stability core of the protein in the near-native intermediate state.

Circular Dichroism↗

PubMed2002

Structure and stability of an acidic fibroblast growth factor from Notophthalmus viridescens.

The three-dimensional solution structure of an acidic fibroblast growth factor (nFGF-1) from the newt (Notophthalmus viridescens) is determined using multidimensional NMR techniques. Complete assignment of all the atoms ((1)H, (15)N, and (13)C) has been achieved using a variety of triple resonance experiments. 50 structures were calculated using hybrid distance geometry-dynamical simulated annealing technique with a total of 1359 constraints. The atomic root mean square distribution for the backbone atoms in the structured region is 0.60 A. The secondary structural elements include 12 beta-strands arranged antiparallely into a beta-barrel structure. The protein (nFGF-1) exists in a monomeric state upon binding to the ligand, sucrose octa sulfate (SOS), in a stoichiometric ratio of 1:1. The SOS binding site consists of a dense cluster of positively charged residues located at the C-terminal end of the molecule. The conformational stabilities of nFGF-1 and its structural and functional homologue from the human source (hFGF-1) are drastically different. The differential stabilities of nFGF-1 and hFGF-1 are attributed to the differences in the number of hydrogen bonds and the presence of solvent inaccessible cavities in the two proteins.

Amino Acid Sequence↗

PubMed2002

Identification of rare partially unfolded states in equilibrium with the native conformation in an all beta-barrel protein.

Human acidic fibroblast growth factor 1 (hFGF-1) is an all beta-barrel protein, and the secondary structural elements in the protein include 12 antiparallel beta-strands arranged into a beta-trefoil fold. In the present study, we investigate the stability of hFGF-1 by hydrogen-deuterium exchange as a function of urea concentration. Urea-induced equilibrium unfolding of hFGF-1 monitored by fluorescence and CD spectroscopy suggests that the protein unfolds by a two-state (native to denatured) mechanism. Hydrogen exchange in hFGF-1, under the experimental conditions used, occurs by the EX2 mechanism. In contrast to the equilibrium unfolding events monitored by optical probes, native state hydrogen exchange data show that the beta-trefoil architecture of hFGF-1 does not behave as a single cooperative unit. There are at least two structurally independent units with differing stabilities in hFGF-1. Beta-strands I, II, III, VI, VII, X, XI, and XII fit into the global unfolding isotherm. By contrast, residues in beta-strands IV, V, VIII, and IX exchange by the subfolding isotherm and could be responsible for the occurrence of high-energy partially unfolded state(s) in hFGF-1. There appears to be a broad continuum of stabilities among the four beta-strands (beta-strands IV, V, VIII, and IX) constituting the subglobal folding unit. The slow exchanging residues in hFGF-1 do not represent the folding nucleus of the protein.

Fibroblast Growth Factor 1↗

PubMed2002

kappa-Hefutoxin1, a novel toxin from the scorpion Heterometrus fulvipes with unique structure and function. Importance of the functional diad in potassium channel selectivity.

An important and exciting challenge in the postgenomic era is to understand the functions of newly discovered proteins based on their structures. The main thrust is to find the common structural motifs that contribute to specific functions. Using this premise, here we report the purification, solution NMR, and functional characterization of a novel class of weak potassium channel toxins from the venom of the scorpion Heterometrus fulvipes. These toxins, kappa-hefutoxin1 and kappa-hefutoxin2, exhibit no homology to any known toxins. NMR studies indicate that kappa-hefutoxin1 adopts a unique three-dimensional fold of two parallel helices linked by two disulfide bridges without any beta-sheets. Based on the presence of the functional diad (Tyr(5)/Lys(19)) at a distance (6.0 +/- 1.0 A) comparable with other potassium channel toxins, we hypothesized its function as a potassium channel toxin. kappa-Hefutoxin 1 not only blocks the voltage-gated K(+)-channels, Kv1.3 and Kv1.2, but also slows the activation kinetics of Kv1.3 currents, a novel feature of kappa-hefutoxin 1, unlike other scorpion toxins, which are considered solely pore blockers. Alanine mutants (Y5A, K19A, and Y5A/K19A) failed to block the channels, indicating the importance of the functional diad.

Amino Acid Sequence↗

PubMed2002

Influence of backbone conformation on protein aggregation.

Effect(s) of organic solvents on an all beta-sheet protein are investigated to understand the influence of backbone conformation on protein aggregation. Results obtained in the present study reveal that protein aggregation is accompanied by the formation of non-native beta-sheet conformation. In contrast, induction of non-native helical segments in the protein is found to inhibit aggregation. The differential effects of the secondary structures on protein aggregation are proposed to stem from the disparity in the nature of the hydrogen bonds and packing of the side chains of hydrophobic residues in the beta-sheet and alpha-helix conformation. In our opinion, the results of the present study provide useful hints to develop methods to alleviate the problems of both in vitro and in vivo protein aggregation.

Animals↗

PubMed2002

Amyloid-like fibril formation in an all beta-barrel protein involves the formation of partially structured intermediate(s).

In the present study, we demonstrate the thermal induced amyloid formation in a beta-barrel protein, such as the acidic fibroblast growth factor from Notopthalmus viridescens (nFGF-1). Fibril formation in nFGF-1 is observed to occur maximally at 65 degrees C. Electron microscope analysis of the thermal induced fibrils of nFGF-1 shows that they are filamentous with an average diameter of about 20 nm. X-ray diffraction analysis reveals that the thermal induced fibrils of nFGF-1 have a typical "cross-beta" structure with the beta-strands perpendicular to the fibril axis. By using a variety of biophysical techniques including multidimensional NMR, we demonstrate that fibril formation involves the formation of a partially structured intermediate(s) in the thermal unfolding pathway of the protein (nFGF-1). Results of the anilino-8-napthalene sulfonate binding experiments indicate that fibril formation occurs due to the coalescence of the protein (in the intermediate state(s)) through the solvent-exposed non-polar surface(s). In this study, we also demonstrate that organic osmolytes, such as proline, can efficiently prevent the thermal induced amyloid formation in nFGF-1. Proline is found to stabilize the native conformation of the protein. The addition, proline is observed to increase the cooperativity of the unfolding (native <--> denatured) reaction and consequently decrease the population of the "sticky" thermal equilibrium intermediate(s) responsible for the fibril formation.

Amyloid↗

PubMed2002

Oligomerization of acidic fibroblast growth factor is not a prerequisite for its cell proliferation activity.

Oligomerization of fibroblast growth factors (FGFs) induced on binding to heparin or heparan sulfate proteoglycan is considered to be crucial for receptor activation and initiation of biological responses. To gain insight into the mechanism of activation of the receptor by FGFs, in the present study we investigate the effect(s) of interaction of a heparin analog, sucrose octasulfate (SOS), on the structure, stability, and biological activities of a recombinant acidic FGF from Notophthalmus viridescens (nFGF-1). SOS is found to bind to nFGF-1 and significantly increase the thermodynamic stability of the protein. Using a variety of techniques such as size-exclusion chromatography, sedimentation velocity, and multidimensional nuclear magnetic resonance (NMR) spectroscopy, it is shown that binding of SOS to nFGF-1 retains the protein in its monomeric state. In its monomeric state (complexed to SOS), n-FGF-1 shows significant cell proliferation activity. (15)N and (1)H chemical shift perturbation and the intermolecular nuclear Overhauser effects (NOEs) between SOS and nFGF-1 reveal that the ligand binds to the dense, positively charged cluster located in the groove enclosed by beta-strands 10 and 11. In addition, molecular modeling based on the NOEs observed for the SOS-nFGF-1 complex, indicates that SOS and heparin share a common binding site on the protein. In conclusion, the results of the present study clearly show that heparin-induced oligomerization of nFGF-1 is not mandatory for its cell proliferation activity.

Animals↗