The complexity of biological membranes presents technical challenges for the analysis of membrane protein biogenesis and function. Here we describe an in vitro fluorescence-based experimental approach for studying the high-resolution structural features of membrane proteins within isolated mitochondria. By this strategy, membrane proteins are cotranslationally labeled with a fluorescent probe at a specific site by the inclusion of aminoacyl tRNA analogs in a cell-free translation system. Labeled proteins are then targeted to the correct subcompartment within active mitochondria by the endogenous import machinery. For each site-specifically labeled protein, a series of rigorous controls must be conducted to ensure the proper membrane integration, topology, and assembly of each labeled sample. The assays described herein serve as the basis for more sophisticated analyses by which multiple fluorescence-based measurements can render detailed information on the topology, microenvironment, and dynamic conformational changes as they occur in real time.
