Identification of rare partially unfolded states in equilibrium with the native conformation in an all beta-barrel protein.
Human acidic fibroblast growth factor 1 (hFGF-1) is an all beta-barrel protein, and the secondary structural elements in the protein include 12 antiparallel beta-strands arranged into a beta-trefoil fold. In the present study, we investigate the stability of hFGF-1 by hydrogen-deuterium exchange as a function of urea concentration. Urea-induced equilibrium unfolding of hFGF-1 monitored by fluorescence and CD spectroscopy suggests that the protein unfolds by a two-state (native to denatured) mechanism. Hydrogen exchange in hFGF-1, under the experimental conditions used, occurs by the EX2 mechanism. In contrast to the equilibrium unfolding events monitored by optical probes, native state hydrogen exchange data show that the beta-trefoil architecture of hFGF-1 does not behave as a single cooperative unit. There are at least two structurally independent units with differing stabilities in hFGF-1. Beta-strands I, II, III, VI, VII, X, XI, and XII fit into the global unfolding isotherm. By contrast, residues in beta-strands IV, V, VIII, and IX exchange by the subfolding isotherm and could be responsible for the occurrence of high-energy partially unfolded state(s) in hFGF-1. There appears to be a broad continuum of stabilities among the four beta-strands (beta-strands IV, V, VIII, and IX) constituting the subglobal folding unit. The slow exchanging residues in hFGF-1 do not represent the folding nucleus of the protein.
Department of Chemistry, National Tsing Hua University, Hsinchu 30013, Taiwan.
Ya-Hui
YH
Chi
Thallampuranam Krishnaswamy S
TK
Kumar
Ing-Ming
IM
Chiu
Chin
C
Yu
D016220:Fibroblast Growth Factor 1 / Q000737:chemistry*; D006801:Humans; D007700:Kinetics; D008958:Models, Molecular; D019906:Nuclear Magnetic Resonance, Biomolecular; D011487:Protein Conformation; D011489:Protein Denaturation; D013816:Thermodynamics
D016220:Fibroblast Growth Factor 1