Susceptibility towards intramolecular disulphide-bond formation affects conformational stability and folding of human basic fibroblast growth factor.
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.
Estapé
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GBF National Research Center for Biotechnology, Biochemical Engineering Division, Mascheroder Weg 1, 38124 Braunschweig, Germany.
van den Heuvel
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J
Rinas
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U
D016475:3T3 Cells / Q000187:drug effects; D000818:Animals; D019256:Cadmium Chloride / Q000737:chemistry; D003545:Cysteine / Q000737:chemistry; D004220:Disulfides / Q000737:chemistry; D016222:Fibroblast Growth Factor 2 / Q000737:chemistry* / Q000494:pharmacology; D006801:Humans; D051379:Mice; D008958:Models, Molecular; D010084:Oxidation-Reduction; D011487:Protein Conformation; D011489:Protein Denaturation; D017510:Protein Folding; D013050:Spectrometry, Fluorescence / Q000379:methods; D014508:Urea / Q000737:chemistry
D004220:Disulfides; D016222:Fibroblast Growth Factor 2; D014508:Urea; D019256:Cadmium Chloride; D003545:Cysteine