RESUMO
Membrane protein structures are highly under-represented relative to water-soluble protein structures in the protein databank. This is especially the case because membrane proteins represent more than 30% of proteins encoded in the human genome yet contribute to less than 10% of currently known structures (Torres et al. in Trends Biol Sci 28:137-144, 2003). Obtaining high-resolution structures of membrane proteins by traditional methods such as NMR and x-ray crystallography is challenging, because membrane proteins are difficult to solubilise, purify and crystallize. Consequently, development of methods to examine protein structure in situ is highly desirable. Fluorescence is highly sensitive to protein structure and dynamics (Lakowicz in Principles of fluorescence spectroscopy, Springer, New York, 2007). This is mainly because of the time a fluorescence probe molecule spends in the excited state. Judicious choice and placement of fluorescent molecule(s) within a protein(s) enables the experimentalist to obtain information at a specific site(s) in the protein (complex) of interest. Moreover, the inherent multi-dimensional nature of fluorescence signals across wavelength, orientation, space and time enables the design of experiments that give direct information on protein structure and dynamics in a biological setting. The purpose of this review is to introduce the reader to approaches to determine oligomeric state or quaternary structure at the cell membrane surface with the ultimate goal of linking the oligomeric state to the biological function. In the first section, we present a brief overview of available methods for determining oligomeric state and compare their advantages and disadvantages. In the second section, we highlight some of the methods developed in our laboratory to address contemporary questions in membrane protein oligomerization. In the third section, we outline our approach to determine the link between protein oligomerization and biological activity.
