Truncated green fluorescent protein (GFP) that is refolded after removing the 10th beta strand can readily bind to a synthetic strand to fully recover the absorbance and fluorescence of the whole protein. This allows rigorous experimental determination of thermodynamic and kinetic parameters of the split system including the equilibrium constant and the association/dissociation rates, which enables residue-specific analysis of peptide-protein interactions. The dissociation rate of the noncovalently-bound strand is observed by strand exchange that is accompanied by a color change, and surprisingly, the rate is greatly enhanced by light irradiation. This peptide-protein photodissociation is a very unusual phenomenon and can potentially be useful for introducing spatially and temporally well-defined perturbations to biological systems as a genetically encoded caged protein.

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-activated cis-trans isomerization 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 accompanying cis-trans isomerization. Comparisons between split GFPs and other photosensory proteins, like photoactive yellow protein and rhodopsin, provide potential strategies for improving the photodissociation quantum yield.split GFP | potential energy surface | photodissociation | cis-trans isomerization | photosensory protein O ptical techniques for investigating biological processes in vivo can achieve subcellular spatial and millisecond temporal resolution by using genetically encoded light-responsive proteins (1). Photoactivatable proteins are used as either imaging tools, such as reversibly photoswitchable fluorescent proteins (RSFPs), with fluorescence that can be modulated by light (2) or optogenetic switches that convert light input into physiological outputs, such as channelrhodopsins, which can regulate ion flow through membranes in response to light (3). Split GFPs have been widely applied for imaging as fluorescent reporters of cellular processes, because they are small (∼25 kDa), are stable in cytosol, produce chromophores autocatalytically, and are amenable to mutation and circular permutation (4). Typically, the protein is expressed as two or more constituent parts linked to separate proteins that only fluoresce on complementing with one another, offering readouts of protein-protein colocalizaton with low background and high specificity (5). However, this technique can generate misleading results, because the GFP complexes, after being formed and fluorescing, are bound irreversibly, which can be highly perturbative to the processes being studied, especially if the protein interaction being probed is ordinarily reversible (6). Although split GFP complexes essentially do not dissocia...

Green fluorescent protein (GFP) variants that carry one extra strand 10 (s10) were created and characterized, and their possible applications were explored. These proteins can fold with either one or the other s10, and the ratio of the two folded forms, unambiguously distinguished by their resulting colors, can be systematically modulated by mutating the residues on s10 or by changing the lengths of the two inserted linker sequences that connect each s10 to the rest of the protein. We have discovered robust empirical rules that accurately predict the product ratios of any given construct in both bacterial and mammalian expressions. Exploiting earlier studies on photo-dissociation of cut s10 from GFP (Do & Boxer, 2011), ratiometric protease sensors were designed from the construct by engineering a specific protease cleavage site into one of the inserted loops, where the bound s10 is replaced by the other strand upon protease cleavage and irradiation with light to switch its color. Since the conversion involves a large spectral shift, these genetically encoded sensors display very high dynamic range. Further engineering of this class of proteins guided by mechanistic understanding of the light-driven process will enable interesting and useful application of the protein.

reporter. Using the second methods we found out that the UCS domain but not the central domain prevents the thermal aggregation of the myosin motor domain. We conclude that while both the UCS domain and the central domain bind the myosin head, only the UCS domain displays chaperone activity. Funded by a grant from the American Heart Association (AHA 13GRNT17290006).

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