Segregation of modified bacteriorhodopsin aggregations in reconstituted vesicle membrane induced by the change of thermodynamical parameters

It was clearly shown that the change in thermodynamical parameters could cause the segregation of membrane protein aggregations in the phospholipid membrane. At first, reconstituted vesicles were prepared with a membrane protein, bacteriorhodopsin and a constituent phospholipid of biomembranes, L-alpha-dimyristoyl phosphatidylcholine. When the temperature of the suspension was decreased or the osmotic pressure was increased by adding poly(ethylene glycol) to this vesicle suspension at 23 degrees, the circular dichroism spectra showed a typical band indicating bacteriorhodopsin trimer formation implying their aggregation. This suggests that the aggregation of trimers proceeded by adding poly(ethylene glycol) into vesicle suspension, just as it proceeded by decreasing the temperature. Next, vesicles were prepared with fluorescein isothiocyanate-labeled bacteriorhodopsin, photoemissive bacteriorhodopsin and L-alpha-dimyristoyl phosphatidylcholine. The excitation energy transfer between the two modified proteins was measured by fluorescence spectroscopy. In this case, however, when poly(ethylene glycol) was added into the suspension, the yield of the excitation energy transfer decreased. This result indicates that modified proteins aggregate separately in a segregated form in the vesicle membrane.