Pressure-exploration of the 33-kDa protein from the spinach photosystem IIparticle

The 33-kDa protein isolated from the spinach photosystem II particle is an ideal model to explore high-pressure protein-unfolding. The protein has a v ery low free energy as previously reported by chemical unfolding studies, s uggesting that it must be easy to modulate its unfolding transition by rath er mild pressure. Moreover, the protein molecule consists of only one trypt ophan residue (Trp241) and eight tyrosine residues, which can be convenient ly used to probe the protein conformation and structural changes under pres sure using either fluorescence spectroscopy or fourth derivative UV absorba nce spectroscopy. The different experimental methods used in the present st udy indicate that at 20 degreesC and pH 6, the 33-kDa protein shows a rever sible two-state unfolding transition from atmospheric pressure to about 180 MPa. This value is much lower than those found for the unfolding of most p roteins studied so far. The unfolding transition induces a large red shift of the maximum fluorescence emission of 34 nm (from 316 nm to 350 nm). The change in standard free energy (DeltaG(o)) and in volume (DeltaV) for the t ransition at pH 6.0 and 20 degreesC are -14.6 kJ.mol(-1) and -120 mL.mol(-1 ), respectively, in which the DeltaG(o) value is consistent with that obtai ned by chemical denaturation. We found that pressure-induced protein unfold ing is promoted by elevated temperatures, which seem largely attributed to the decrease in the absolute value of DeltaG(o) (only a minor variation was observed for the DeltaV value). However, the promotion of the unfolding by alkaline pH seems mainly related to the increase in DeltaV without any sig nificant changes in DeltaG(o). It was also found that NaCl significantly pr otects the protein from pressure-induced unfolding. In the presence of 1 m NaCl, the pressure needed to induce the half-unfold of the protein is shift ed to a higher value (shift of 75 MPa) in comparison with that observed wit hout NaCl. Interestingly, in the presence of NaCl, the value of DeltaV is s ignificantly reduced whilst that of DeltaG(o) remains as before. The unfold ing-refolding kinetics of the protein has also been studied by pressure-jum p, in which it was revealed that both reactions are a two-state transition process with a relatively slow relaxation time of about 10(2) s.