Proteins perform most cellular functions in macromolecular complexes. Quantitative information on macromolecular complexes is scarce. The same protein often participates in different complexes to exhibit diverse functionality. Current ensemble approaches of identifying cellular protein interactions cannot reveal physiological permutations of these interactions. I will discuss a single-molecule pull-down (SiMPull) assay that combines the principles of a conventional pull-down assay with single-molecule fluorescence microscopy and enables direct visualization of individual cellular protein complexes. SiMPull can reveal how many proteins and of which kinds are present in the in vivo complex, as we show using protein kinase A. We then demonstrate a wide applicability to various signaling proteins found in the cytosol, membrane and cellular organelles, and to endogenous protein complexes from animal tissue extracts. The pulled-down proteins are functional and are used, without further processing, for single-molecule biochemical studies. SiMPull should provide a rapid, sensitive and robust platform for analyzing protein assemblies in biological pathways.
Yang Kevin Xiang's lab focuses on the function of a family of GPCRs (adrenergic receptors) in both heart and neurodegeneration diseases (Alzheimer and Parkinson). GPCRs are nature's most versatile biological sensors responding to hormones and neurotransmitters, and mediate the senses of pressure, sight, smell, and taste. Adrenergic receptors (ARs), which transmit signals in both central and peripheral nerve systems, are one of the most extensively characterized and serve as a model system for understanding the structure, cell biology, and physiology of GPCRs. His lab uses in vitro and in vivo systems to determine the structural and cellular basis for more complex functional properties that are only observed in differentiated cells and tissues.
Researchers should cite this work as follows:
1000 MNTL, University of Illinois, Urbana-Champaign, Urbana, IL