title: Mechanism and bottlenecks in strand photodissociation of split green fluorescent proteins (GFPs).
abstract: Split GFPs have been widely applied for monitoring protein-protein interactions by expressing GFPs as two or more constituent parts linked to separate proteins that only fluoresce on complementing with one another. Although this complementation is typically irreversible, it has been shown previously that light accelerates dissociation of a noncovalently attached β-strand from a circularly permuted split GFP, allowing the interaction to be reversible. Reversible complementation is desirable, but photodissociation has too low of an efficiency (quantum yield <1%) to be useful as an optogenetic tool. Understanding the physical origins of this low efficiency can provide strategies to improve it. We elucidated the mechanism of strand photodissociation by measuring the dependence of its rate on light intensity and point mutations. The results show that strand photodissociation is a two-step process involving light-activatedcis-transisomerization of the chromophore followed by light-independent strand dissociation. The dependence of the rate on temperature was then used to establish a potential energy surface (PES) diagram along the photodissociation reaction coordinate. The resulting energetics-function model reveals the rate-limiting process to be the transition from the electronic excited-state to the ground-state PES accompanyingcis-transisomerization. Comparisons between split GFPs and other photosensory proteins, like photoactive yellow protein and rhodopsin, provide potential strategies for improving the photodissociation quantum yield.
authors.affiliation: Department of Chemistry, Stanford University, Stanford, CA 94305-5012.
authors.forename: Chi-Yun
authors.initials: CY
authors.lastname: Lin
authors.affiliation: Department of Chemistry, Stanford University, Stanford, CA 94305-5012.
authors.forename: Johan
authors.initials: J
authors.lastname: Both
authors.affiliation: Department of Chemistry, Stanford University, Stanford, CA 94305-5012.
authors.forename: Keunbong
authors.initials: K
authors.lastname: Do
authors.affiliation: Department of Chemistry, Stanford University, Stanford, CA 94305-5012 sboxer@stanford.edu.
authors.forename: Steven G
authors.initials: SG
authors.lastname: Boxer
mesh_terms: D000465:Algorithms; D015870:Gene Expression; D049452:Green Fluorescent Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D007536:Isomerism; D008958:Models, Molecular; D008962:Models, Theoretical; D057054:Molecular Imaging; D009154:Mutation; D010777:Photochemistry; D011485:Protein Binding; D011487:Protein Conformation; D025941:Protein Interaction Mapping; D013329:Structure-Activity Relationship
chemical_list: D049452:Green Fluorescent Proteins
keywords: cis-trans isomerization; photodissociation; photosensory protein; potential energy surface; split GFP