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Green Fluorescent Protein

Green fluorescent protein (GFP) and related fluorescent proteins have become an integral part of molecular biology for a variety of uses including visualizing protein localization and as intracellular sensors (i.e. pH, Ca2+, redox environment).  The widespread use of GFP is owed to its remarkable self-catalyzed chromophore formation. We have worked on GFP photophysics and photochemistry since the beginning of its application in imaging, in particular study the mechanism of excited state proton transfer. [136]


Split GFPs are proteins that have been separated into two polypeptides that together compose the entire primary sequence of GFP.  Many split GFPs that do not spontaneously reassemble into a GFP-like structure can be reassembled by fusing both halves of GFP to interacting proteins.  Because the split GFPs require fused interacting proteins to form the chromophore, the primary use of split GFPs is for imaging protein-protein interactions in vivo.

Although split GFPs are widely used for imaging in vivo, little is known about the reassembly mechanism.  We have developed a method to remove single structural elements of split GFPs and replace them with synthetic peptides, which allow enormous flexibility in sequence and the ability to incorporate unnatural amino acids. [259], [267]  We aim to use this highly controlled system to better understand the biophysics of split GFP reconstitution in vitro in order to inform experiments using split GFP systems in vivo.  We are also interested in using our synthetic methods for producing fluorescent proteins with novel photophysical properties, as well as better understanding the photochemistry of GFP. In addition to protein engineering, we use ultrafast fluorescence and a host of other analytical methods to characterize these novel proteins.

  Recent Publications      

"Synthetic Control of Green Fluorescent Protein", Kevin P. Kent, Luke M. Oltrogge, Steven G. Boxer, Journal of the American Chemical Society, in press (2009). [pdf] [Supplement]

"Deconstructing Green Fluorescent Protein", Kevin Kent, William Childs, and Steven G. Boxer, J. Am. Chem. Soc., 130, 9664-9665, (2008). [pdf] [Supplement]

The Boxer LaboratoryStanford UniversityDepartment of Chemistry • 380 Roth Way, Stanford, California, 94305-5012 • (650) 723-4482
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