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

Thiagarajan Srimathi ^*, Thallampuranam Krishnaswamy S. Kumar , Karuppanan Muthusamy Kathir ^*, Ya-Hui Chi ^*, Sampath Srisailam ^*, Wann-Yin Lin , Ing-Ming Chiu  and Chin Yu 

^*Department of Chemistry, National Tsing Hua University, Hsinchu, Taiwan; Department of Chemistry and Biochemistry, University of Arkansas, Fayetteville, Arkansas 72701; Department of Chemistry, National Taiwan University, Taipei, Taiwan; and Department of Internal Medicine, The Ohio State University, Columbus, Ohio 43210

Correspondence: Address reprint requests to Chin Yu, Dept. of Chemistry and Biochemistry, University of Arkansas, Fayetteville, AR 72701. Tel.: 479-575-2724; Fax: 479-575-4049; E-mail: cyu{at}uark.edu.

Acidic fibroblast growth factors from human (hFGF-1) and newt (nFGF-1) (Notopthalamus viridescens) are 16-kDa, all ß-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.