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

• Published in: Biophysical journal 2003-07, Vol.85 (1), p.459-472
• Main Authors: Srimathi, Thiagarajan, Kumar, Thallampuranam Krishnaswamy S., Kathir, Karuppanan Muthusamy, Chi, Ya-Hui, Srisailam, Sampath, Lin, Wann-Yin, Chiu, Ing-Ming, Yu, Chin
• Format: Article
• Language: English
• Subjects: Amino Acid Sequence; Animals; Chromatography, Gel; Deuterium Exchange Measurement; Fibroblast Growth Factor 1 - chemistry; Fibroblast Growth Factor 1 - classification; Humans; Magnetic Resonance Spectroscopy; Molecular Sequence Data; Notophthalmus viridescens; Protein Conformation; Protein Denaturation; Protein Folding; Protein Isoforms - chemistry; Protein Isoforms - classification; Proteins; Sequence Homology, Amino Acid; Species Specificity; Spectrometry, Fluorescence; Structure-Activity Relationship
• ISSN: 0006-3495; 1542-0086
• DOI: 10.1016/S0006-3495(03)74491-9

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.