Time-dependent changes in the denatured state(s) influence the folding mechanism of an all beta-sheet protein.
Newt fibroblast growth factor (nFGF-1) is an approximately 15-kDa all beta-sheet protein devoid of disulfide bonds. Urea-induced equilibrium unfolding of nFGF-1, monitored by steady state fluorescence and far-UV circular dichroism spectroscopy, is cooperative with no detectable intermediate(s). Urea-induced unfolding of nFGF-1 is reversible, but the percentage of the protein recovered in the native state depends on the time of incubation of the protein in the denaturant. The yield of the protein in the native state decreases with the increase in time of incubation in the denaturant. The failure of the protein to refold to its native state is not due to trivial chemical reactions that could possibly occur upon prolonged incubation in the denaturant. (1)H-(15)N heteronuclear single quantum coherence (HSQC) spectra, limited proteolytic digestion, and fluorescence data suggest that the misfolded state(s) of nFGF-1 has structural features resembling that of the denatured state(s). GroEL, in the presence of ATP, is observed to rescue the protein from being trapped in the misfolded state(s). (1)H-(15)N HSQC data of nFGF-1, acquired in the denatured state(s) (in 8 m urea), suggest that the protein undergoes subtle time-dependent structural changes in the denaturant. To our knowledge, this report for the first time demonstrates that the commitment to adapt unproductive pathways leading to protein misfolding/aggregation occurs in the denatured state ensemble.
Kathir
Karuppanan Muthusamy
KM
Department of Chemistry and Biochemistry, University of Arkansas, Fayetteville, 72701, USA.
Kumar
Thallapuranam Krishnaswamy S
TK
Rajalingam
Dakshinamurthy
D
Yu
Chin
C
D000818:Animals; D018834:Chaperonin 60 / Q000494:pharmacology; D016220:Fibroblast Growth Factor 1 / Q000737:chemistry*; D011489:Protein Denaturation; D017510:Protein Folding; D017433:Protein Structure, Secondary; D012455:Salamandridae; D013997:Time Factors; D014508:Urea / Q000494:pharmacology
D018834:Chaperonin 60; D016220:Fibroblast Growth Factor 1; D014508:Urea