Identification of Rare Partially Unfolded States in Equilibrium with the Native Conformation in an All β-Barrel Protein

Abstract

Human acidic fibroblast growth factor 1 (hFGF-1) is an all ␤-barrel protein, and the secondary structural elements in the protein include 12 antiparallel ␤-strands arranged into a ␤-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 ␤-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. ␤-Strands I, II, III, VI, VII, X, XI, and XII fit into the global unfolding isotherm. By contrast, residues in ␤-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 ␤-strands (␤-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.

Detection of Folding Sites of β-Trefoil Fold Proteins Based on Amino Acid Sequence Analyses and Structure-Based Sequence Alignment

“…It has been reported that this functional site folds in the late stage of the folding process (the foldability-function tradeoff hypothesis) [13,14,21]. In preceding studies, residues in β-strand 2 and β-strands 5-8 are protected in the early stage of folding revealed by H/D exchange experiments of NMR [17,18]. Longo et al [13][14] and Xia et al [15] reported that the folding nucleus detected by the ϕ-value analyses consists of residues 16-58 in β-strands 2-6.…”

“…simulations [20,21] for FGF-1, IL-1β and for His were performed to identify their folding mechanisms. These studies revealed the differences in overall folding pathways among these proteins, while these proteins commonly start to fold at the central β-strands [13,17].…”

GuHCl and NaCl-dependent Hydrogen Exchange in MerP Reveals a Well-defined Core with an Unusual Exchange Pattern

“…7,9 However, in the presence of denaturant at high concentrations, the amide proton may start to exchange by the EX1 mechanism where k rc [k cl . 9 To investigate whether the assumption of EX2 exchange is valid at the highest concentration of GuHCl used here, we calculated the rate constant for the folding and unfolding of MerP at 1 M GuHCl, using the parameters gained from the chevron plot previously measured for MerP.…”

“…23,24 For amide protons that gave free energy of exchange versus [GuHCl] plots with upward curvature, the curve was fitted to equation (7) according to the denaturant binding model described by Tanford, 25,26 which considers the differential binding of denaturant to a limited number of sites:…”

Structure and Stability of an Acidic Fibroblast Growth Factor from Notophthalmus viridescens

“…The high affinity receptors are composed of an extracellular ligand-binding domain consisting of three immunoglobulin (Ig)-like domains, a transmembrane domain, and a cytoplasmic tyrosine kinase domain (9 -12). The low affinity receptors have been identified as heparin sulfate proteoglycans (13)(14)(15)(16)(17)(18)(19). Although the significance of binding to these polyanions is not yet clear, heparin/heparan sulfate are believed to be required for binding of FGF-1 to their high affinity receptor (20 -23).…”