Green Fluorescent Protein (GFP)

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 depends on its remarkable self-catalyzed chromophore formation. We have worked on GFP photophysics and photochemistry since the beginning of its application in imaging, in particular to study general features of charge transfer, proton transfer and electrostatic control of chromophore isomerization, the latter a focus of current work (136, 163, 249, 250, 302, 306).

 

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 [325].  By using split GFPs, we have developed tools to interrogate the photophysics and photochemistry of fluorescent proteins with versatile synthetic control, allowing for enormous flexibility in sequence and the ability to incorporate non-canonical amino acids (259, 267). We discovered several unique light-dependent properties, such as photodissociation and photoassociation of split GFP fragments (273, 280, 319), opening an avenue for a wide range of optogenetic applications along with imaging capabilities (296). We are currently aiming to optimize this system through directed evolution and by obtaining a deeper understanding of the underlying mechanism (314). 

[330]  "Structural Evidence of Photoisomerization Pathways in Fluorescent Proteins”, Jeffrey Chang, Matthew G. Romei, and Steven G. Boxer, Journal of the American Chemical Society, 141, 15504-15508 (2019)  [pdf​]

[329] "Unified Model for Photophysical and Electro-Optical Properties of Green Fluorescent Proteins", Chi-Yun Lin, Matthew G. Romei, Luke M. Oltrogge, Irimpan I. Mathews, and Steven G. Boxer, Journal of the American Chemical Society, 141, 15250-15265 (2019) [pdf]

[326]  “Split Green Fluorescent Proteins (GFPs): Scope, Limitations, and Outlook”, Matthew G. Romei and Steven G. Boxer, Annual Reviews of Biophysics, 48, 19-44 (2019) [pdf]

[319]  "Structural Insight into the Photochemistry of Split Green Fluorescent Proteins: A Unique Role for a His-tag", Alan Deng and Steven G. Boxer, Journal of the American Chemical Society, 140, 375–381(2018). [pdf]

 

[314]  "Mechanism and bottlenecks in strand photodissociation of split green fluorescent proteins (GFPs)", Chi-Yun Lin (林其勻), Johan Both, Keunbong Do, and Steven G. Boxer, PNAS, Published online, E2146–E2155 (2017). [pdf]

The Boxer Laboratory, Stanford University, Department of Chemistry, Stanford, CA, 94305-5012

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