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. Study holds ProTherm entries: 16671, 16672, 16673, 16674 Extra Details: two-state; stable intermediate; molten globule; structural interactions
Submitter: Connie Wang
Submission Date: April 24, 2018, 8:48 p.m.
|Number of data points||12|
|Proteins||Fibroblast growth factor 1 ; Fibroblast growth factor 1 ; Fibroblast growth factor 1 ; Fibroblast growth factor 1|
|Assays/Quantities/Protocols||Experimental Assay: Cm prot_conc:50 microM ; Experimental Assay: m prot_conc:50 microM ; Experimental Assay: dG_H2O prot_conc:50 microM ; Experimental Assay: Cm prot_conc:100 microg/ml ; Experimental Assay: m prot_conc:100 microg/ml ; Experimental Assay: dG_H2O prot_conc:100 microg/ml|
|Libraries||Mutations for sequence QKPKLLYCSNGGYFLRIFPDGKVDGTRDRSDPYIQLQFYAESVGEVYIKSLETGQYLAMDSDGQLYASQSPSEECLFLERLEENNYNTYKSKVHADKDWFVGIKKNGKTKPGSRTHFGQKAILFLPLPVSSD ; Mutations for sequence FNLPPGNYKKPKLLYCSNGGHFLRILPDGTVDGTRDRSDQHIQLQLSAESVGEVYIKSTETGQYLAMDTDGLLYGSQTPNEECLFLERLEENHYNTYISKKHAEKNWFVGLKKNGSCKRGPRTHYGQKAILFLPLPVSSD|