Susceptibility towards intramolecular disulphide-bond formation affects conformational stability and folding of human basic fibroblast growth factor

Abstract

The conformational stability and the folding properties of the all-beta-type protein human basic fibroblast growth factor (hFGF-2) were studied by means of fluorescence spectroscopy. The results show that the instability of the biological activity of hFGF-2 is also reflected in a low conformational stability of the molecule. The reversibility of the unfolding and refolding process was established under reducing conditions. Determination of the free-energy of unfolding in the presence of reducing agents revealed that the conformational stability of hFGF-2 (DeltaGH2Oapp congruent with21 kJ. mol-1, 25 degreesC) is low compared with other globular proteins under physiological conditions (20-60 kJ.mol-1). However, the conformational stability of hFGF-2 is particularly low under non-reducing conditions. This instability is attributed to intramolecular disulphide-bond formation, rendering the molecule more susceptible to denaturant-induced unfolding. In addition, denaturant-induced unfolding of hFGF-2 renders the protein more susceptible to irreversible oxidative denaturation. Experimental evidence is provided that the irreversibility of the unfolding and refolding process in the absence of reducing agents is linked to the formation of an intramolecular disulphide bond involving cysteines 96 and 101.

Inclusion body anatomy and functioning of chaperone-mediated in vivo inclusion body disassembly during high-level recombinant protein production in Escherichia coli

“…Native hFGF-2 purified to homogeneity from the soluble cell fraction (Seeger and Rinas, 1996) was subjected to increasing concentrations of the chaotropic agent guanidinium hydrochloride (Gdn-HCl) for unfolding studies under reducing conditions (Estapé et al, 1998; (Estapé et al, 1998). In comparison, aggregates of hFGF-2 generated in vitro by subjecting native hFGF-2 to 70°C or recovered as in vivo produced inclusion bodies using the temperature-inducible expression system were also subjected to increasing concentrations of Gdn-HCl under reducing conditions ( Fig.…”

“…Unfolding was monitored by measurement of the protein fluorescence emission at 355 nm (excitation at 280 nm) as described previously (Estapé et al, 1998).…”

“…Guanidine-hydrochloride (Gdn-HCl) induced unfolding studies of native hFGF-2 based on changes in fluorescent properties were performed as described (Estapé et al, 1998). 20…”

“…hFGF-2 is a protein of 155 amino acids, 5 sufficiently small to be incorporated into the central cavity formed by the two stacked homoheptameric GroEL rings (Sakikawa et al, 1999;Houry et al, 1999). It is a protein of low stability, in particular under non-reducing conditions (Estapé et al, 1998) and exhibits very slow folding kinetics (Estapé and Rinas, 1999). Temperature-induced production of hFGF-2 initially leads to the accumulation of hFGF-2 exclusively in the aggregated form 10 (Hoffmann and Rinas, 2000).…”

“…Soluble and insoluble fractions were obtained after subsequent centrifugation at 15,000 g for one hour. The amount of solubilized hFGF-2 was determined by fluorescence emission at 355 nm (excitation 280 nm) as described previously (Estapé et al, 1998). 10…”