Structurally Homologous All β-Barrel Proteins Adopt Different Mechanisms of Folding

AbstractAcidic fibroblast growth factors from human (hFGF-1) and newt (nFGF-1) (Notopthalamus viridescens) are 16-kDa, all β-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 0.96M GdnHCl. The intermediate (in hFGF-1) populated maximally at 0.96M 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

A method for the prevention of thrombin-induced degradation of recombinant proteins

A new strategy to prevent degradation of recombinant proteins caused by non-specific cleavage by thrombin is described. We demonstrate that degradation due to non-specific cleavage of recombinant protein mediated by thrombin can be completely prevented by separation of thrombin from the recombinant protein on spin columns packed with heparin-sepharose. This method is generally applicable to all recombinant proteins that require the thrombin for the cleavage of affinity tags for purification. To our knowledge, this is the first report of an efficient and reliable method for the separation of residual thrombin from purified recombinant proteins

Structurally Homologous All β-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 β-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

Solution structure of the ligand binding domain of the fibroblast growth factor receptor : Role of heparin in the activation of the receptor

The three-dimensional solution Structure of the ligand binding D2 domain of the fibroblast growth factor receptor (FGFR) is determined using Multidimensional NMR techniques. The atomic root mean-square distribution for the backbone atoms in the structured region is 0.64 angstrom. Secondary structural elements in the D2 domain include beta-strands arranged antiparallely into two layers of beta-sheets. The Structure of the D2 domain is characterized by the presence of a short flexible helix that protrudes Out Of the layers of beta-sheets. Results of size exclusion chromatography and sedimentation velocity experiments show that the D2 domain exists in a monomeric state both in the presence and in the absence of bound,sucrose octasulfate (SOS), a Structural analogue of heparin. Comparison of the Solution structure of the D2 domain with the crystal structure of the protein (D2 domain) in the FGF signaling complex reveals significant differences, suggesting that ligand (FGF) binding may induce significant conformational changes in the receptor. SOS binding sites in the D2 domain have been mapped on the basis of the H-1-N-15 chemical shift perturbation data. SOS binds to the positively charged residues located in beta-strand III and the flexible helix. isothermal titration calorimetry data indicate that the ligand (hFGF-1) binds strongly (K-d similar to 10(-9) M) to the D2 domain even in the absence of SOS. Binding of SOS to either the D2 domain or hFGF-1 does not seem to be the driving force for the formation of the D2-hFGF-1 binary complex. The function of SOS binding appears to stabilize the preformed D2-FGF binary complexNRC publication: Ye