A partly Folded State of Acidic Fibroblast Growth Factor at Low Ph

Abstract

Acid denaturation of acidic fibroblast growth factor (aFGF) at low ionic strength was monitored by far-ultraviolet circular dichroism and intrinsic fluorescence. The two spectroscopic probes displayed noncoincident transitions, which suggested the accumulation of partly folded species around pH 4.0. Although under these conditions the fluorescence of aFGF resembled that of the unfolded form of the protein, farultraviolet circular dichroism and proton nuclear magnetic resonance spectra indicated the presence of persistent secondary and tertiary structure. Moreover, at pH 4.0, aFGF showed cooperative thermal denaturation and interacted weakly with the hydrophobic probe N-phenyl-1-naphthylamine, showing a relatively high level of structure that did not fit into the classical molten globule category. This intermediate is also capable of interacting with liposomes and might represent a membrane translocation-competent form.Keywords: protein structure ; protein folding ; folding intermediate; translocation across membranes.Acidic and basic fibroblast growth factors (aFGF and bFGF, respectively) are proteins that induce mitogenesis in most mesoderm-and neuroectoderm-derived cell lines, and show many other hormone-like activities. The three-dimensional structures of the two polypeptides have been determined by X-ray diffraction and 'H-NMR [ l , 21. Their folding topology is similar and follows the p-trefoil motif [3]. However, no clear-cut differences in the biological activities between these two proteins have been observed [4-61. Both bind with high affinity to heparin and they are also referred to as heparin-binding growth factors. The interaction with heparin protects FCF proteins against denaturation [4] and is necessary for their binding to transmembrane tyrosine kinase receptors on the membrane to trigger mitogenesis [7, 81. Binding of FGF to their receptors causes tyrosine phosphorylation of several proteins, including the receptor, and translocation of the protein to the cell nucleus [9, 101 where it induces DNA synthesis. The study of the process of FGF translocation is, therefore, of special interest. It has been shown that aFGF fused to diphteria toxin translocates to the cytosol by a mechanism requiring low pH (4.3, and this process is prevented by the presence of heparin, which stabilizes FGF against pH denaturation [11-131. Recently, it has been reported that low pH also induces interaction of aFGF with acidic phospholipid membranes [ 141. Thus, it seems that translocation-competent aFGF has a partially folded structure like that induced by moderately acidic pH conditions. There is ample evidence that Correspondence to J. M. Sanz, Centro de Investigaciones Biologicas Fax: +34 15649075.

Unfolding Studies of Escherichia coli Maltodextrin Glucosidase Monitored by Fluorescence Spectroscopy

“…It was observed that the ANS fluorescence emission reached the highest value at 0.25 M GdnHCl and then gradually decreased to a lowest value during unfolding of MalZ (Fig. 5a and b a significant portion of their hydrophobic cores to the solvent [13]. Hence, ANS binds strongly to the MG state of proteins and fluoresces intensely.…”

“…ANS has been immensely useful in the identification of equilibrium intermediates such as the molten-globule state (MG). Molten-globule-like intermediates usually display a significant exposure of hydrophobic cores to the solvent [13]. Hence, ANS binds strongly to MG state and fluoresces intensely.…”

15N NMR Relaxation Studies of Free and Ligand-bound Human Acidic Fibroblast Growth Factor

“…In contrast, results of other studies suggest a protective role for heparin/heparan sulfate (12)(13)(14). Binding of hFGF to the glycosaminoglycans is shown to protect FGFs against proteolytic digestion and heat-and acid-induced unfolding (15)(16)(17).…”

“…One useful method to study motion of protein molecules involves the analysis of NMR relaxation processes (19,20). With the advent of inverse detection methods, it has been feasible to analyze the backbone dynamics of proteins at a residue level using 15 N relaxation measurements (21)(22)(23)(24)(25)(26) 4 Cl was purchased from Cambridge Isotope Laboratories. SOS was purchased from Toronto Research Chemicals.…”

“…15 N longitudinal (R 1 ), transverse (R 2 ) relaxation rates and { 1 H}-15 N steadystate nuclear Overhauser effects were obtained at 500 and 600 MHz (at 25°C) for all resolved backbone amide groups using 1 H-detected two-dimensional NMR experiments. Relaxation data were fit to the extended model free dynamics for each NH group.…”