Protein Folding / De-folding Study

The folding/unfolding of 23kDa protein from spinach photosystem II (P23k) and recombinant porcine insulin precursor (rPIP) were studied in this thesis using high hydrostatic pressure,various spectroscopies as well as many other methods.In the research of P23K, pressure-induced unfolding of P23k from spinach photosystem II has been systematically investigated at various experimental conditions. Thermodynamic equilibrium studies indicate that the protein is very sensitive to pressure. At 20 oC and pH 5.5, P23k was unfolded completely under pressure near 180 MPa, which is much lower than most natural proteins studied to date. The free energy (?Gu) and volume change (?Vu) for the unfolding are 5.6 kcal/mol and–150.3 ml/mol respectively. It was found NaCl and sucrose significantly stabilize the protein from unfolding. It was interesting that the mechanisim of stabilization is related not only with an increase in the free energy caused by the increase of surface tension around the protein molecules but also with the reduction in absolute value of the volume change upon unfolding. The pressure-jump studies of P23k reveal a negative activation volume for unfolding (- 66.2 ml/mol) and a positive activation volume for refolding (84.1 ml/mol), indicating that in terms of system volume, the protein transition state lies between the folded and unfolded states. Examination of the temperature effect on the unfolding kinetics indicates that the thermal expansibility of the transition state and the unfolded state of P23k are closer each other and they are larger than that of the native state. The diverse pressure-refolding pathways of P23k in some conditions were revealed in pressure-jump kinetics. In the research of rPIP, it was found that bis-ANS can bind to the hydrophobic areas of rPIP and human insulin. And the fluorescence changes of bis-ANS were related with the conformation changes of the two molecules during the folding/unfolding process. Based on this observation, we used bis-ANS as an extrinsic fluorescence probe to explore folding/unfolding of rPIP in detail and then compared the results with human insulin. The time cause of bis-ANS fluorescence change upon rPIP refolding obtained from trapping method and dynamic method both indicated that formations of three disulfide bridges were ordinal. And Qiao's results also supported this conclusion. We also found that the refolding rate of rPIP obtained from stopped-flow kinetics was 149s much faster than that of human insulin (16700s). It is interesting that we found a rapid process in dynamic measurement which companied with the fluorescence of bis-ANS rapidly increasing and then decreasing after the starting of rPIP refolding, whereas the similar process can not be observed in the research of human insulin. The unfolding of rPIP caused by disulfide bond reduction was also examined using bis-ANS fluorescence by thermal and dynamic measurement. The results indicated the three disulfides reduction of rPIP was in turn. During the disulfides breaking of human insulin, we also found the similar results.