Enumeration of oligomerization states of membrane proteins in living cells by homo-FRET spectroscopy and microscopy: theory and application.
Protein-protein interactions play a pivotal role in biological signaling networks. It is highly desirable to perform experiments that can directly assess the oligomerization state and degree of oligomerization of biological macromolecules in their native environment. Homo-FRET depends on the inverse sixth power of separation between interacting like fluorophores on the nanometer scale and is therefore sensitive to protein oligomerization. Homo-FRET is normally detected by steady-state or time-resolved fluorescence anisotropy measurements. Here we show by theory and simulation that an examination of the extent of homotransfer as measured by steady-state fluorescence anisotropy as a function of fluorophore labeling (or photodepletion) gives valuable information on the oligomerization state of self-associating proteins. We examine random distributions of monomers, dilute solutions of oligomers, and concentrated solutions of oligomers. The theory is applied to literature data on band 3 protein dimers in membranes, GPI-linked protein trimers in "rafts," and clustered GFP-tagged epidermal growth factor receptors in cell membranes to illustrate the general utility and applicability of our analytical approach.
Yeow
Edwin K L
EK
Division of Chemistry and Biological Chemistry, School of Physical and Mathematical Sciences, Nanyang Technological University, Singapore 637616.
Clayton
Andrew H A
AH
D000465:Algorithms; D002468:Cell Physiological Phenomena; D003198:Computer Simulation; D019281:Dimerization; D031541:Fluorescence Resonance Energy Transfer / Q000379:methods*; D008565:Membrane Proteins / Q000737:chemistry* / Q000378:metabolism* / Q000648:ultrastructure; D008856:Microscopy, Fluorescence / Q000379:methods*; D008954:Models, Biological; D008956:Models, Chemical; D008958:Models, Molecular; D046912:Multiprotein Complexes / Q000737:chemistry / Q000378:metabolism / Q000648:ultrastructure; D025941:Protein Interaction Mapping / Q000379:methods*
D008565:Membrane Proteins; D046912:Multiprotein Complexes