Structure and Photoreaction of Photoactive Yellow Protein, a Structural Prototype of the PAS Domain Superfamily†

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...

Section: Resultsmentioning

confidence: 67%

Mechanism and bottlenecks in strand photodissociation of split green fluorescent proteins (GFPs)

Lin

Both

et al. 2017

“…Importantly, the analysis readily distinguishes a LOV domain from its most closely related protein domains, which include non-LOV PAS domains (including PYP, "photoactive yellow protein") and other flavoproteins, including BLUF domain photoreceptors ("Blue-Light Using FAD") (18,26,(31)(32)(33) (Fig. 2C and Dataset S1).…”

Section: Resultsmentioning

confidence: 99%

Functional and topological diversity of LOV domain photoreceptors

Glantz

Carpenter

Melkonian

et al. 2016

132

155

Light-oxygen-voltage sensitive (LOV) flavoproteins are ubiquitous photoreceptors that mediate responses to environmental cues. Photosensory inputs are transduced into signaling outputs via structural rearrangements in sensor domains that consequently modulate the activity of an effector domain or multidomain clusters. Establishing the diversity in effector function and sensor-effector topology will inform what signaling mechanisms govern light-responsive behaviors across multiple kingdoms of life and how these signals are transduced. Here, we report the bioinformatics identification of over 6,700 candidate LOV domains (including over 4,000 previously unidentified sequences from plants and protists), and insights from their annotations for ontological function and structural arrangements. Motif analysis identified the sensors from ∼42 million ORFs, with strong statistical separation from other flavoproteins and non-LOV members of the structurally related Per-aryl hydrocarbon receptor nuclear translocator (ARNT)-Sim family. Conserved-domain analysis determined putative light-regulated function and multidomain topologies. We found that for certain effectors, sensor-effector linker length is discretized based on both phylogeny and the preservation of α-helical heptad repeats within an extended coiled-coil linker structure. This finding suggests that preserving sensor-effector orientation is a key determinant of linker length, in addition to ancestry, in LOV signaling structure-function. We found a surprisingly high prevalence of effectors with functions previously thought to be rare among LOV proteins, such as regulators of G protein signaling, and discovered several previously unidentified effectors, such as lipases. This work highlights the value of applying genomic and transcriptomic technologies to diverse organisms to capture the structural and functional variation in photosensory proteins that are vastly important in adaptation, photobiology, and optogenetics.photoreceptors | LOV | flavoproteins | optogenetics

“…Absorption of a photon triggers the isomerization of the chromophore and the subsequent thermal reaction cycle (13,14). The hydrogen-bonding network near the chromophore is modulated during the thermal reaction, resulting in proton transfers within the network that are associated with large conformational changes (15)(16)(17)(18). Two SHBs are formed between pCA and E46 and between pCA and Y42.…”

mentioning

confidence: 99%

Low-barrier hydrogen bond in photoactive yellow protein

Yamaguchi

Kamikubo

Kurihara

et al. 2009

Self Cite

194

363

Low-barrier hydrogen bonds (LBHBs) have been proposed to play roles in protein functions, including enzymatic catalysis and proton transfer. Transient formation of LBHBs is expected to stabilize specific reaction intermediates. However, based on experimental results and theoretical considerations, arguments against the importance of LBHB in proteins have been raised. The discrepancy is caused by the absence of direct identification of the hydrogen atom position. Here, we show by high-resolution neutron crystallography of photoactive yellow protein (PYP) that a LBHB exists in a protein, even in the ground state. We identified Ϸ87% (819/942) of the hydrogen positions in PYP and demonstrated that the hydrogen bond between the chromophore and E46 is a LBHB. This LBHB stabilizes an isolated electric charge buried in the hydrophobic environment of the protein interior. We propose that in the excited state the fast relaxation of the LBHB into a normal hydrogen bond is the trigger for photo-signal propagation to the protein moiety. These results give insights into the novel roles of LBHBs and the mechanism of the formation of LBHBs.neutron crystallography ͉ photoreaction ͉ proton translocation ͉ short hydrogen bond T he idea that the formation of low-barrier hydrogen bonds (LBHBs) plays an essential role in enzyme catalysis was proposed in the early 1990s (1, 2). Although several lines of circumstantial evidence support the existence of LBHBs, negative results have also been published (3-5). This discrepancy is caused by the absence of direct demonstration of LBHBs in proteins. In general, hydrogen bonds in proteins are identified by the distance between a donor and an acceptor within the crystal structure. Because of its abnormally short bond length, a LBHB is accompanied by a quasi-covalent bond feature, whereas an ordinary hydrogen bond can be depicted as an electrostatic interaction between a donor-proton dipole and a dipole (or a monopole) on an acceptor atom (6-8). In LBHBs, the proton is shared by the donor and acceptor atoms, resulting in the distribution of the hydrogen between the two (6). Therefore, to identify a LBHB, it is essential to determine the position of the hydrogen atom and those of the donor and acceptor atoms. Recently, it was shown that a light sensor protein, photoactive yellow protein (PYP), contains 2 short hydrogen bonds (SHBs) adjacent to the reaction center, even in the ground state (9, 10). The hydrogen atoms involved in the SHBs, however, could not be observed either by X-ray crystallography at atomic resolution (9, 11) or neutron crystallography at 2.5-Å resolution (10).PYP is a putative photoreceptor for negative phototaxis of the purple phototropic bacterium, Halorhodospira halophila (12). The chromophore of PYP, p-coumaric acid (pCA), is buried in a hydrophobic pocket. Absorption of a photon triggers the isomerization of the chromophore and the subsequent thermal reaction cycle (13,14). The hydrogen-bonding network near the chromophore is modulated during the thermal reaction, result...