Immobilization in surface-tethered lipid vesicles as a new tool for singlebiomolecule spectroscopy

Single-molecule fluorescence studies of functional biomolecule dynamics rel y on the ability to provide biologically relevant experimental conditions. Long measurement times on single molecules require their immobilization, wh ich might modify their dynamics through interactions with the trapping medi um, e.g., a class surface or a polymer gel. In an effort to to overcome thi s problem we have devised a new immobilization technique, based on the conf inement of single biomolecules inside 100 nm surface-tethered lipid vesicle s. The number of molecules in each vesicle can be accurately determined fro m fluorescence time traces; under our experimental conditions the number di stribution of encapsulated molecules obeys a Poisson distribution with an a verage occupancy of 0.65 molecules per vesicle. It is further shown that th e distribution of fluorescence polarization values of trapped molecules can serve as a sensitive probe for their freedom of motion and thus for the en vironment they sample inside the liposomes. Polarization distributions are obtained for two vesicle-entrapped labeled proteins, bovine serum albumin a nd adenylate kinase, and compared with distributions measured for the same proteins directly adsorbed on glass. From the significant relative narrowin g of the distributions for encapsulated molecules, it is concluded that the ir motion within the vesicles is quite similar to free solution.