SUSCEPTIBILITY TOWARDS INTRAMOLECULAR DISULFIDE-BOND FORMATION AFFECTS CONFORMATIONAL STABILITY AND FOLDING OF HUMAN BASIC FIBROBLAST-GROWTH-FACTOR

The conformational stability and the folding properties of the all-bet a-type protein human basic fibroblast growth factor (hFGF-2) were stud ied by means of fluorescence spectroscopy. The results show that the i nstability of the biological activity of hFGF-2 is also reflected in a low conformational stability of the molecule. The reversibility of th e unfolding and refolding process was established under reducing condi tions. Determination of the free-energy of unfolding in the presence o f reducing agents revealed that the conformational stability of hFGF-2 (Delta G(app)(H2O) congruent to 21 kJ.mol-(1,) 25 degrees C) is low c ompared with other globular proteins under physiological conditions (2 0-60 kJ.mol(-1)). However, the conformational stability of hFGF-2 is p articularly low under non-reducing conditions. This instability is att ributed to intramolecular disulphide-bond formation, rendering the mol ecule more susceptible to denaturant-induced unfolding. In addition, d enaturant-induced unfolding of hFGF-2 renders the protein more suscept ible to irreversible oxidative denaturation. Experimental evidence is provided that the irreversibility of the unfolding and refolding proce ss in the absence of reducing agents is linked to the formation of an intramolecular disulphide bond involving cysteines 96 and 101.