SINGLE MOLECULAR ASSAY OF INDIVIDUAL ATP TURNOVER BY A MYOSIN-GFP FUSION PROTEIN EXPRESSED IN-VITRO

Fusion proteins of a truncated mutant of myosin subfragment-1 (S1dC) a nd green fluorescent protein (GFP) were expressed in vitro by T7 RNA p olymerase and rabbit reticulocyte lysate, Single S1dC-GFP fusion prote ins were clearly seen and their individual ATP turnovers were directly monitored using low background total internal reflection fluorescence microscopy (LBTIRFM), recently developed by our laboratory, LBTIRFM u sing GFP as a fluorescent tag allowed us to assay functions of single protein molecules expressed in vitro, Thus, the results suggested that this method may be particularly useful to analyze functions of protei ns that cannot be produced in an active form and/or in large quantitie s in conventional heterologous expression systems. (C) 1997 Federation of European Biochemical Societies.