Understanding the Folding Pathway(s) of a Beta-Barrel Protein

 

                  T. K. S. Kumar

Department of Chemistry, National Tsing Hua University

                 Hsinchu, TAIWAN

 

Human acidic fibroblast growth factor (hFGF-1) is a 15 kDa, all beta-sheet protein, bereft of disulfide bonds.  The secondary structural elements in the protein include twelve beta strands arranged antiparallely into a beta barrel structure.  Equilibrium unfolding experiments on hFGF-1 revealed that the protein accumulates in an intermediate state in 0.96 M guanidinium hydrochloride (GdnHCl). Chemical shift perturbation and hydrogen deuterium exchange experiments monitored by 1H-15N HSQC showed that profound structural changes in the intermediate state (in 0.96 M GdnHCl) occurs in the C-terminal, heparin binding region of the protein.

 

        The chronology of events occurring in the refolding pathway of hFGF-1 has been investigated using a variety of techniques such as stopped-flow fluorescence, stopped-flow circular dichroism, and quenched-flow hydrogen-deuterium exchange in conjunction with multidimensional NMR spectroscopy.  Measurement of the unfolding and refolding rates in various concentrations of urea revealed that the refolding of hFGF-1 proceeds through accumulation of kinetic intermediates.  Results of the quench-flow hydrogen exchange showed that the hydrogen bonds linking the N- and C-terminal ends are the first to form during the refolding of hFGF-1. We believe that some of the kinetic events observed in the refolding pathway of hFGF-1 could be common to other beta barrel proteins.