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-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...

Split Green Fluorescent Proteins: Scope, Limitations, and Outlook

“…Initial results showed that the photodissociation rate rises linearly at low light intensity before plateauing, suggesting a two-step mechanism in which the first is light dependent and the second is a thermal process (26, 78). Further mechanistic insight came from studying both nonphotoswitchable and photoswitchable (E222Q mutant) circular permutants, which revealed that the light-dependent step is cis-trans isomerization of the chromophore followed by thermal strand dissociation (78). These findings link the properties of photodissociable split GFPs to reversibly photoswitchable GFPs that have been extensively engineered for super-resolution microscopy.…”

“…Further information on the energy landscape was obtained from the temperature dependence of photoactivation, strand dissociation, and fluorescence, with results summarized compactly in Figure 9 (78). The first steps in this mechanism are similar to those found for other protein-based photoisomerizable systems such as rhodopsin and photoactive yellow protein.…”

“…( b ) Topology of GFP’s 11 β-strands and internal α-helix (ih). Figure adapted with permission from Reference 78.…”

“…Branching points are shown as circled numbers and are color coded to match their associated processes: ❶ represents the excited-state barrier partitioning fluorescence and isomerization; ❷ represents the photochemical funnel, which divides aborted from successful isomerization; and ❸ represents branching of strand dissociation and thermal relaxation. Abbreviations: C, noncovalently bound fluorescent protein complex with a cis chromophore in the ground electronic state; C’, noncovalently bound fluorescent protein complex with a trans chromophore in the ground electronic state; D, truncated protein containing a trans chromophore in the ground electronic state with the dissociated strand removed; P, dissociated strand (peptide); γ, population branching ratio at the photochemical funnel; E a,diss , energy barrier for thermal strand dissociation; E a,fwd , energy barrier in the excited-state to reach photochemical funnel; FC, Franck-Condon excitation from the ground- to the excited-state; k BC , rate constant for excited-state barrier crossing; k diss , rate constant for strand dissociation; k FI , rate constant for fluorescence; k IC , rate constant for nonradiative internal conversion without passing through the photochemical funnel; k th , rate constant for thermal relaxation from C’ to C. Figure adapted with permission from Reference 78.…”

Development and Applications of Superfolder and Split Fluorescent Protein Detection Systems in Biology

“…Split-GFP association is a slow but irreversible process. Dissociation of split-GFP fragments can only be achieved in vitro by photodissociation of the reconstituted barrel [112] or chemical disruption of peptide bonds in denaturation conditions [113]. Renaturation of isolated split-GFP fragments induces the reassociation of the two fragments into a functional GFP molecule, with a five-time increase in fluorescence intensity [114,115].…”