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SummaryRpBphP2 and RpBphP3, two tandem bacteriophytochromes from the photosynthetic bacterium Rhodopseudomonas palustris, share high sequence identity but exhibit distinct photoconversion behavior. Unlike the canonical RpBphP2, RpBphP3 photoconverts to an unusual near-red-absorbing (Pnr) state; both are required for synthesis of light-harvesting complexes under low-light conditions. Here we report the crystal structures of the photosensory core modules of RpBphP2 and RpBphP3. Despite different quaternary structures, RpBphP2 and RpBphP3 adopt nearly identical tertiary structures. The RpBphP3 structure reveals tongue-and-groove interactions at the interface between the GAF and PHY domains. A single mutation in the PRxSF motif at the GAF-PHY interface abolishes light-induced formation of the Pnr state in RpBphP3, possibly due to altered structural rigidity of the chromophore-binding pocket. Structural comparisons suggest that long-range signaling involves structural rearrangement of the helical spine at the dimer interface. These structures, together with mutational studies, provide insights into photoconversion and the long-range signaling mechanism in phytochromes.

Absorption of a photon in the visible region of the solar spectrum is the fundamental step in the photochemical reactions that drive biological processes such as imaging, photosynthesis, and sensory perception. All known photoreceptors are proteins that contain a chromophore: a small organic molecule that absorbs in the visible region of the spectrum ( 1 ). The bacterial integral membrane protein bacteriorhodopsin (bR) uses retinal as its chromophore (see the figure), capturing the energy in a photon to drive retinal isomerization and pump protons across the membrane. bR is a readily accessible analog to the visual rhodopsins in, for example, the human eye. On page 145 of this issue, Nogly et al. ( 2 ) used time-resolved x-ray crystallography ( 3 , 4 ) to determine intermediate structures of bR at near-atomic resolution on the femtosecond to picosecond time scales at which isomerization occurs.

Summary Aureochrome1, a signaling photoreceptor from a eukaryotic photosynthetic stramenopile, confers blue-light-regulated DNA binding on the organism. Its topology, in which a C-terminal LOV sensor domain is linked to an N-terminal DNA-binding bZIP effector domain, contrasts with the reverse sensor-effector topology in most other known LOV-photoreceptors. How, then, is signal transmitted in Aureochrome1? The dark- and light-state crystal structures of Aureochrome1 LOV domain (AuLOV) show that its helical N- and C-terminal flanking regions are packed against the external surface of the core β sheet, opposite to the FMN chromophore on the internal surface. Light-induced conformational changes occur in the quaternary structure of the AuLOV dimer and in Phe298 of the Hβ strand in the core. The properties of AuLOV extend the applicability of LOV domains as versatile design modules that permit fusion to effector domains via either the N- or C-termini to confer blue-light sensitivity.

Cellular processes and indeed the survival of entire organisms crucially depend on precise spatiotemporal coordination of a multitude of molecular events. A new tool in cell biology is denoted "optogenetics" which describes the use of genetically encoded, light-gated proteins, i.e. photoreceptors, which perturb and control cellular and organismal behavior in a spatiotemporally exact manner. Photoreceptors resemble fluorescent reporter proteins such as GFP in being genetically encoded, non-invasive, and applicable to intact cells and organisms. They are explicitly intended to modulate activity; in contrast, fluorescent proteins generally do not disturb the processes under study. Fluorescent proteins have revolutionized cell biology because they allow the monitoring of such processes by imaging techniques that offer superb spatiotemporal resolution and sensitivity. Optogenetics extends these advantages to offer control. The scope of optogenetics has recently been expanded beyond the use of naturally occurring photoreceptors by the biologically-inspired design of engineered (or synthetic) photoreceptors. These photoreceptors are derived by fusion of one or more light-absorbing sensor domains with an output or effector domain displaying the activity to be controlled. Here, we focus on the design and application of such engineered photoreceptors. We treat basic signaling principles and discuss the two photosensor classes which are currently most widely used in fusion-based design: LOV domains and phytochromes. Based on these principles, we develop general strategies for the engineering of photoreceptors. Finally, we review recently successful examples of the design and application of engineered photoreceptors. Our perspective provides guidelines for researchers interested in developing and applying novel optogenetic tools.

Survival of organisms in dynamic environments requires accurate perception and integration of signals. At the molecular level, signal detection is mediated by signal receptor proteins that largely are of modular composition. Sensor modules, such as the widespread Per–ARNT–Sim (PAS) domains, detect signals and, in response, regulate the biological activity of effector modules. Here, we exploit the modularity of signal receptors to design and engineer synthetic receptors that comprise two PAS sensor domains responsive to different signals, and we use these signals to control the activity of a histidine kinase effector. Designed two-input PAS receptors detected oxygen and blue light in a positive cooperative manner. The extent of the response to the signals was dictated by domain topology: the dominant regulatory effect was exerted by the PAS domain proximal to the effector domain. The presence of one sensor domain modulated the signal response function of the other. Sequence and structural data on natural receptors with tandem PAS domains show that these are predominantly linked by short amphipathic α-helices. Signals from multiple sensor domains could be integrated and propagated to the effector domain as torques. Our results inform the rational design of receptors that integrate multiple signals to modulate cellular behavior.

Signaling photoreceptors use the information contained in the absorption of a photon to modulate biological activity in plants and a wide range of organisms. The fundamental—and as yet imperfectly answered—question is, how is this achieved at the molecular level? We adopt the perspective of biophysicists interested in light-dependent signal transduction in nature and the three-dimensional structures that underpin signaling. Six classes of photoreceptors are known: light-oxygen-voltage (LOV) sensors, xanthopsins, phytochromes, blue-light sensors using flavin adenine dinucleotide (BLUF), cryptochromes, and rhodopsins. All are water-soluble proteins except rhodopsins, which are integral membrane proteins; all are based on a modular architecture except cryptochromes and rhodopsins; and each displays a distinct, light-dependent chemical process based on the photochemistry of their nonprotein chromophore, such as isomerization about a double bond (xanthopsins, phytochromes, and rhodopsins), formation or rupture of a covalent bond (LOV sensors), or electron transfer (BLUF sensors and cryptochromes).

Phytochromes are red-light photoreceptors that regulate light responses in plants, fungi, and bacteria via reversible photoconversion between red (Pr) and far-red (Pfr) light-absorbing states. Here we report the crystal structure at 2.9 Å resolution of a bacteriophytochrome from Pseudomonas aeruginosa with an intact, fully photoactive photosensory core domain in its dark-adapted Pfr state. This structure reveals how unusual interdomain interactions, including a knot and an “arm” structure near the chromophore site, bring together the PAS (Per-ARNT-Sim), GAF (cGMP phosphodiesterase/adenyl cyclase/FhlA), and PHY (phytochrome) domains to achieve Pr/Pfr photoconversion. The PAS, GAF, and PHY domains have topologic elements in common and may have a single evolutionary origin. We identify key interactions that stabilize the chromophore in the Pfr state and provide structural and mutational evidence to support the essential role of the PHY domain in efficient Pr/Pfr photoconversion. We also identify a pair of conserved residues that may undergo concerted conformational changes during photoconversion. Modeling of the full-length bacteriophytochrome structure, including its output histidine kinase domain, suggests how local structural changes originating in the photosensory domain modulate interactions between long, cross-domain signaling helices at the dimer interface and are transmitted to the spatially distant effector domain, thereby regulating its histidine kinase activity.

Light sensing by photoreceptors controls phototropism, chloroplast movement, stomatal opening, and leaf expansion in plants. Understanding the molecular mechanism by which these processes are regulated requires a quantitative description of photoreceptor dynamics. We focus on a light-driven signal transduction mechanism in the LOV2 domain (LOV, light, oxygen, voltage) of the blue light photoreceptor phototropin 1 from Avena sativa (oat). High-resolution crystal structures of the dark and light states of an oat LOV2 construct including residues Leu404 through Leu546 (LOV2 (404-546)) have been determined at 105 and 293 K. In all four structures, LOV2 (404-546) exhibits the typical Per-ARNT-Sim (PAS) fold, flanked by an additional conserved N-terminal turn-helix-turn motif and a C-terminal flanking region containing an amphipathic Jalpha helix. These regions dock on the LOV2 core domain and bury several hydrophobic residues of the central beta-sheet of the core domain that would otherwise be exposed to solvent. Light structures of LOV2 (404-546) reveal that formation of the covalent bond between Cys450 and the C4a atom of the flavin mononucleotide (FMN) results in local rearrangement of the hydrogen-bonding network in the FMN binding pocket. These rearrangements are associated with disruption of the Asn414-Asp515 hydrogen bond on the surface of the protein and displacement of the N- and C-terminal flanking regions of LOV2 (404-546), both of which constitute a structural signal.

Bacteriophytochromes RpBphP2 and RpBphP3 from the photosynthetic bacterium Rhodopseudomonas palustris work in tandem to modulate synthesis of the light-harvesting complex LH4 in response to light. Although RpBphP2 and RpBphP3 share the same domain structure with 52% sequence identity, they demonstrate distinct photoconversion behaviors. RpBphP2 exhibits the “classical” phytochrome behavior of reversible photoconversion between red (Pr) and far-red (Pfr) light-absorbing states, whereas RpBphP3 exhibits novel photoconversion between Pr and a near-red (Pnr) light-absorbing states. We have determined the crystal structure at 2.2-Å resolution of the chromophore binding domains of RpBphP3, covalently bound with chromophore biliverdin IXα. By combining structural and sequence analyses with site-directed mutagenesis, we identify key residues that directly modulate the photochemical properties of RpBphP3 and RpBphP2. Remarkably, we identify a region spanning residues 207–212 in RpBphP3, in which a single mutation, L207Y, causes this unusual bacteriophytochrome to revert to the classical phenotype that undergoes reversible photoconversion between the Pr and Pfr states. The reverse mutation, Y193L, in the corresponding region in RpBphP2 significantly diminishes the formation of the Pfr state. We propose that residues 207–212 and the spatially adjacent conserved residues, Asp-216 and Tyr-272, interact with the chromophore and form part of the interface between the chromophore binding domains and the PHY domain that modulates photoconversion.

Two-component signal transduction is the major signaling mechanism that enables bacteria to survive and thrive in complex environmental conditions. The photosynthetic bacterium R. palustris employs two tandem bacteriophytochromes, RpBphP2 and RpBphP3, to perceive red-light signals that regulate the synthesis of light-harvesting complexes under low-light conditions. Both RpBphP2 and RpBphP3 are photosensory histidine kinases coupled to the same response regulator RPA3017. Together, they constitute a two-component system that converts a red-light signal into a biological signal. In this work, the crystal structure of RPA3017 in the unphosphorylated form at 1.9 Å resolution is presented. This structure reveals a tightly associated dimer arrangement that is conserved among phytochrome-related response regulators. The conserved active-site architecture provides structural insight into the phosphotransfer reaction between RpBphP2/RpBphP3 and RPA3017. Based on structural comparisons and homology modeling, how specific recognition between RpBphP2/RpBphP3 and RPA3017 is achieved at the molecular level is further explored.

The flavin-binding BLUF domain of AppA represents a new class of blue light photoreceptors that are present in a number of bacterial and algal species. The dark state X-ray structure of this domain was determined at 2.3 A resolution. The domain demonstrates a new function for the common ferredoxin-like fold; two long alpha-helices flank the flavin, which is bound with its isoalloxazine ring perpendicular to a five-stranded beta-sheet. The hydrogen bond network and the overall protein topology of the BLUF domain (but not its sequence) bear some resemblance to LOV domains, a subset of PAS domains widely involved in signaling. Nearly all residues conserved in BLUF domains surround the flavin chromophore, many of which are involved in an intricate hydrogen bond network. Photoactivation may induce a rearrangement in this network via reorientation of the Gln63 side chain to form a new hydrogen bond to the flavin O4 position. This shift would also break a hydrogen bond to the Trp104 side chain, which may be critical in induction of global structural change in AppA.

Photoactive yellow protein (E-PYP) is a blue light photoreceptor, implicated in a negative phototactic response in Ectothiorhodospira halophila , that also serves as a model for the Per–Arnt–Sim superfamily of signaling molecules. Because no biological signaling partner for E-PYP has been identified, it has not been possible to correlate any of its photocycle intermediates with a relevant signaling state. However, the PYP domain (Ppr-PYP) from the sensor histidine kinase Ppr in Rhodospirillum centenum , which regulates the catalytic activity of Ppr by blue light absorption, may allow such issues to be addressed. Here we report the crystal structure of Ppr-PYP at 2 Å resolution. This domain has the same absorption spectrum and similar photocycle kinetics as full length Ppr, but a blue-shifted absorbance and considerably slower photocycle than E-PYP. Although the overall fold of Ppr-PYP resembles that of E-PYP, a novel conformation of the β4–β5 loop results in inaccessibility of Met-100, thought to catalyze chromophore reisomerization, to the chromophore. This conformation also exposes a highly conserved molecular surface that could interact with downstream signaling partners. Other structural differences in the α3–α4 and β4–β5 loops are consistent with these regions playing significant roles in the control of photocycle dynamics and, by comparison to other sensory Per–Arnt–Sim domains, in signal transduction. Because of its direct linkage to a measurable biological output, Ppr-PYP serves as an excellent system for understanding how changes in photocycle dynamics affect signaling by PYPs.

The phototropins constitute an important class of plant photoreceptor kinases that control a range of physiological responses, including phototropism, light-directed chloroplast movement, and light-induced stomatal opening. The LOV2 domain of phototropin binds a molecule of flavin mononucleotide (FMN) and undergoes a photocycle involving light-driven covalent adduct formation between a conserved cysteine residue and the C(4a) atom of FMN. This product state promotes C-terminal kinase activation and downstream signal transduction. Here, we report the primary photophysics and photochemistry of LOV2 domains of phototropin 1 of Avena sativa (oat) and of the phy3 photoreceptor of Adiantum capillus-veneris (maidenhair fern). In agreement with earlier reports [Swartz, T. E., et al. (2001) J. Biol. Chem. 276, 36493-36500], we find that the FMN triplet state is the reactive species from which the photoreaction occurs. We demonstrate that the triplet state is the primary photoproduct in the LOV2 photocycle, generated at 60% efficiency. No spectroscopically distinguishable intermediates precede the FMN triplet on the femtosecond to nanosecond time scale, indicating that it is formed directly via intersystem crossing (ISC) from the singlet state. Our results indicate that the majority of the FMN triplets in the LOV2 domain exist in the protonated form. We propose a reaction mechanism that involves excited-state proton transfer, on the nanosecond time scale or faster, from the sulfhydryl group of the conserved cysteine to the N5 atom of FMN. This event promotes adduct formation by increasing the electrophilicity of C(4a) and subsequent nucleophilic attack by the cysteine's thiolate anion. Comparison to free FMN in solution shows that the protein environment of LOV2 increases the ISC rate of FMN by a factor of 2.4, thus improving the yield of the cysteinyl-flavin adduct and the efficiency of phototropin-mediated signaling processes.

Phototropin, a major blue-light receptor for phototropism in seed plants, exhibits blue-light-dependent autophosphorylation and contains two light, oxygen, or voltage (LOV) domains and a serine/threonine kinase domain. The LOV domains share homology with the PER-ARNT-SIM (PAS) superfamily, a diverse group of sensor proteins. Each LOV domain noncovalently binds a single FMN molecule and exhibits reversible photochemistry in vitro when expressed separately or in tandem. We have determined the crystal structure of the LOV2 domain from the phototropin segment of the chimeric fern photoreceptor phy3 to 2.7-Å resolution. The structure constitutes an FMN-binding fold that reveals how the flavin cofactor is embedded in the protein. The single LOV2 cysteine residue is located 4.2 Å from flavin atom C(4a), consistent with a model in which absorption of blue light induces formation of a covalent cysteinyl-C(4a) adduct. Residues that interact with FMN in the phototropin segment of the chimeric fern photoreceptor (phy3) LOV2 are conserved in LOV domains from phototropin of other plant species and from three proteins involved in the regulation of circadian rhythms in Arabidopsis and Neurospora . This conservation suggests that these domains exhibit the same overall fold and share a common mechanism for flavin binding and light-induced signaling.

A key role in signal transduction and dimerization mediated by Per–Arnt–Sim (PAS) domains is played by α-helical linkers that flank the structurally similar α/β cores of these domains. However, crystal-packing forces and the different construct lengths and sequences of the PAS domains influence the final length and orientation of the linkers relative to the core and create uncertainty in the exact mechanism of the linker function. Thus, structural characterization and comparison of the linkers within isolated PAS-domain constructs and/or full-length PAS-containing proteins is important for clarification of the mechanism. The plant blue-light photoreceptors phototropins possess two N-terminal flavin mononucleotide-based light, oxygen or voltage (LOV) domains (LOV1 and LOV2) that comprise a subclass of the PAS family and one C-terminal serine/threonine kinase domain whose enzymatic activity is regulated by blue light. The dark-adapted state crystal structures of the Arabidopsis thaliana phototropin 1 and phototropin 2 LOV1-domain constructs flanked by an N-terminal A′α helix and the structure of the phototropin 2 core LOV2 domain are known. Here, the crystal structure of the A. thaliana phototropin 1 LOV2 domain has been determined in its dark-adapted state. The core is flanked by an N-terminal A′α helix and a C-terminal Jα helix similar to those in the previously reported structure of Avena sativa phototropin 1 LOV2. In contrast to the monomeric A. sativa LOV2, A. thaliana LOV2 is a dimer in which two A′α helices adopt a scissor-like orientation at the dimer interface and form a short α-helical coiled coil. The Jα helix predominantly interacts with the β-sheet and plays a role in coiled-coil formation and dimerization.

Heme oxygenase (HO) catalyzes the degradation of heme to biliverdin, ferrous iron, and carbon monoxide (CO) using reducing equivalents and molecular oxygen (O2) (1–3). The major physiological roles of HO in mammals are the recycling of iron, defense against oxidative stress, and the generation of CO as a signal transmitter. Although CO is generally toxic, a small amount of CO is proposed to be involved in physiological processes such as anti-inflammation, anti-apoptosis, anti-proliferation, and vasodilation (4). The HO reaction proceeds via a multi-step mechanism and continuous availability of O2 is important for the overall reaction (Figure 1). Three molecules of O2 are required for a complete reaction cycle, which occurs without release of the reaction intermediates derived from heme. Because CO preferentially binds to the ferrous heme iron and competes with binding of O2, CO produced during the HO reaction is potentially a powerful inhibitor. The reaction intermediates shown in Figure 1 do not dissociate from HO during the reaction, which means that CO produced with verdoheme may still be present at the subsequent reaction step. If so, CO could compete with the binding of O2 to ferrous verdoheme, the next step in conversion of verdoheme to biliverdin-iron chelate (5). Indeed, CO-bound verdoheme can be observed spectroscopically under normal, single turnover reaction conditions in vitro (6, 7). To avoid potential product inhibition by CO, HO must possess a much more stringent mechanism to discriminate between CO and O2 than in other heme proteins. Thus, the ratio of the affinities of CO to O2 for rat HO-1 in complex with heme (heme-rHO-1) is between 1.2 and 5.6 (8), in contrast with much larger ratios of 41 and 95 for myoglobin (Mb) (9) and leghemoglobin (10), respectively. Figure 1 Reaction scheme of HO. The overall HO reaction proceeds via a multi-step mechanism. The first step is the oxidation of heme to α-hydroxyheme, requiring O2 and reducing equivalents supplied by NADPH cytochrome P450 reductase. O2 bound to the heme ... The discrimination mechanism between CO and O2 in globins has been well studied. Two mechanisms have been proposed: increase in the affinity for O2 by the formation of hydrogen bonds or increase of the polarity in the heme pocket, and a steric inhibitory effect on CO binding (9). In both Mb and hemoglobin (Hb), the distal histidine is located close to the ligand binding site. This histidine forms a hydrogen bond with O2 bound to the heme iron, and thereby stabilizes O2 binding. In addition, an early X-ray crystallographic study (11) showed that the Fe-CO bond angle in Mb is strongly bent, presumably by steric hindrance between CO and the distal histidine, which destabilizes CO binding. However, more recent ultra-high-resolution X-ray crystallographic (12), spectroscopic (13), and systematic biochemical (9) studies are inconsistent with the hypothesis of steric hindrance in globins. On the other hand, steric hindrance has been recently proposed to discriminate between O2 and CO binding in bacterial cytochrome c’, based on crystal structures and an electrochemical study (14), and in the heme binding domain of FixL, based on ultrafast infrared spectroscopy following CO photolysis (15). Thus, the mechanism of discrimination may differ among heme proteins. We previously determined crystal structures of heme-rHO-1 complexed with several ligands and demonstrated that upon CO or cyanide binding, the heme and the proximal A-helix slide along the α-γ axis of heme, and the distal F-helix slides in the opposite direction (16). These conformational changes do not occur upon nitric oxide (NO) or azide binding. We proposed that this conformational change is specific for ligands which prefer a linear binding geometry such as CO and cyanide because the distal atom of these ligands would collide with the carbonyl group of Gly-139 if the ligands adopt a linear binding geometry. No such collision would occur for ligands which prefer a bent binding geometry such as NO and azide. Because O2 also prefers a bent binding geometry, we expect that the conformational changes would not occur upon O2 binding. The conformational change upon CO binding is not restricted to mammalian HO-1. A similar change has been reported in one bacterial HO homolog (17) but was absent in another (18). Steric hindrance thus seems to be one of the mechanisms for discrimination between CO and O2 in HO. However, neither the O2-bound heme-rHO-1 structure nor the extent of structural constraints in the CO-bound and O2-bound forms have been examined. In order to probe further details of the discrimination mechanism in HO, we have determined the O2-bound heme-rHO-1 structure, which reveals that the conformational changes are specific to CO binding. We also demonstrate that the conformational changes in heme-rHO-1 upon CO photolysis at cryogenic temperature ~100 K are the reverse of those upon CO binding. We previously determined the CO-photolyzed structure at ~35 K, but could not detect any conformational changes upon photolysis (with the limited exception of some water movement and CO trapping at two specific sites) because all protein motions are severely restricted at ~35 K (19). We therefore repeated our CO photolysis experiments at ~100 K, a temperature where we anticipated that more extensive conformational changes could occur. Our new structural data clearly show the reverse conformational changes at the kinked part of the distal F-helix. This implies that CO binding in heme-rHO-1 is severely constrained compared to that in Mb (20–23) and in the fully-liganded, relaxed state (R-state) of Hb (24), it may be similarly constrained to the fully-unliganded, tense state (T-state) of Hb (24). We also observe a putative CO-trapping site that suggests a new pathway for CO migration in heme-rHO-1. However, we do not observe the reverse conformational changes in the proximal A-helix, though the heme iron does move. A recent time-resolved resonance Raman study that follows CO photolysis in heme-rHO-1 (25) has shown behavior in the ν(Fe-His) stretching mode specific to HO. We discuss the relationship between spectroscopic and crystallographic results.

X-ray free electron lasers (XFELs) are potentially revolutionary X-ray sources because of their very short pulse duration, extreme peak brilliance and high spatial coherence, features that distinguish them from today's synchrotron sources. We review recent time-resolved Laue diffraction and time-resolved wide angle X-ray scattering (WAXS) studies at synchrotron sources, and initial static studies at XFELs. XFELs have the potential to transform the field of time-resolved structural biology, yet many challenges arise in devising and adapting hardware, experimental design and data analysis strategies to exploit their unusual properties. Despite these challenges, we are confident that XFEL sources are poised to shed new light on ultrafast protein reaction dynamics.

Signaling photoreceptors mediate diverse organismal adaptations in response to light. As light-gated protein switches, signaling photoreceptors provide the basis for optogenetics, a term that refers to the control of organismal physiology and behavior by light. We establish as novel optogenetic tools the plasmids pDusk and pDawn, which employ blue-light photoreceptors to confer light-repressed or light-induced gene expression in Escherichia coli with up to 460-fold induction upon illumination. Key features of these systems are low background activity, high dynamic range, spatial control on the 20-μm scale, independence from exogenous factors, and ease of use. In optogenetic experiments, pDusk and pDawn can be used to specifically perturb individual nodes of signaling networks and interrogate their role. On the preparative scale, pDawn can induce by light the production of recombinant proteins and thus represents a cost-effective and readily automated alternative to conventional induction systems.

Various organisms can sense light through a large family of signaling proteins known as photoreceptors. Upon absorption of a photon in the appropriate wavelength range, photoreceptors undergo structural changes in the chromophore, an organic pigment embedded in the photosensory module of the protein. Phytochromes are red-light photoreceptors originally discovered in plants and more recently in bacteria. They are unique in their ability to undergo reversible photoconversion between two photoisomerizable states, Pr (red light ∼ 700 nm) and Pfr (far-red light ∼ 750 nm). In the photosynthetic bacterium, Rhodopseudomonas palustris, a pair of bacteriophytochromes, RpBphP2 (P2) and RpBphP3 (P3) modulate synthesis of a light-harvesting complex. P2 and P3 have the same biliverdin chromophore (BV) and share 52% amino acid sequence identity, yet they have distinct photoconversion properties. P2, similar to classical bacteriophytochromes, alternates between Pr and Pfr states. P3 is unusual since it alternates between Pr and a unique Pnr (near-red light ∼ 650 nm) state. Our experimental goal is to identify amino acid residues that form specific interactions with BV during photoconversion and as such play key roles in forming distinct photoisomerizable states of P2 and P3. Through site-directed mutagenesis of conserved amino acids informed by structural and sequence analysis of bacteriophytochromes, we created a single-amino acid mutant variant of P3 T480P that undergoes classic reversible photoconversion between Pr and Pfr states. We also report on P2 and P3 mutants that show minimal absorption in the red region of visible light spectrum or are naturally more fluorescent than wild-type proteins. Currently, we are investigating chemical mechanisms that justify observed P2 and P3 mutant phenotypes.

We describe here the results obtained to date on a project aimed at characterizing the changes occurring in the protein product (p21) of the H-ras proto-oncogene during and as a result of hydrolysis of GTP at its active site. The approach used involves crystallization of p21 with a photosensitive precursor of GTP (caged GTP) at the active site followed by generation of GTP by photolysis and collection of X-ray diffraction data using the Laue method at a synchrotron source. The structure of p21 complexed with a single diastereomer of caged GTP is presented here. In contrast to crystals obtained with mixed diastereomers, the nucleotide appears to bind in a manner which is very similar to that of other guanine nucleotides (GDP, GTP, GppNHp). The current state of time resolved structural experiments using these crystals is presented.

Phytochromes are red-light photoreceptors that regulate light responses in plants, fungi, and bacteria by means of reversible photoconversion between red (Pr) and far-red (Pfr) light-absorbing states. Here, we report the crystal structure of the Q188L mutant of Pseudomonas aeruginosa bacteriophytochrome (PaBphP) photosensory core module, which exhibits altered photoconversion behavior and different crystal packing from wild type. We observe two distinct chromophore conformations in the Q188L crystal structure that we identify with the Pfr and Pr states. The Pr/Pfr compositions, varying from crystal to crystal, seem to correlate with light conditions under which the Q188L crystals are cryoprotected. We also compare all known Pr and Pfr structures. Using site-directed mutagenesis, we identify residues that are involved in stabilizing the 15 E a (Pfr) and 15 Z a (Pr) configurations of the biliverdin chromophore. Specifically, Ser-261 appears to be essential to form a stable Pr state in PaBphP, possibly by means of its interaction with the propionate group of ring C. We propose a “flip-and-rotate” model that summarizes the major conformational differences between the Pr and Pfr states of the chromophore and its binding pocket.

Per-ARNT-Sim (PAS) domains serve as widely-distributed, versatile, sensor and interaction modules in signal transduction proteins. PAS sensors detect a wide range of chemical and physical stimuli and regulate the activity of functionally diverse effector domains. In contrast to this chemical, physical and functional diversity, the structure of the core of PAS domains is broadly conserved and comprises a five-stranded antiparallel β-sheet and several α-helices. Signals originate within the conserved core and generate structural and dynamic changes predominantly within the β-sheet, from which they propagate via amphipathic α-helical and coiled-coil linkers at the N- or C-termini of the core to the covalently-attached effector domain. Effector domains are typically dimeric; their activity appears to be largely regulated by signal-dependent changes in quaternary structure and dynamics. The signaling mechanisms of PAS and other signaling domains share common features, and these commonalities can be exploited to enable structure-based design of artificial photo- and chemosensors.

Signal transduction proteins are organized into sensor (input) domains that perceive a signal and, in response, regulate the biological activity of effector (output) domains. We reprogrammed the input signal specificity of a normally oxygen-sensitive, light-inert histidine kinase by replacing its chemosensor domain by a light-oxygen-voltage photosensor domain. Illumination of the resultant fusion kinase YF1 reduced net kinase activity by approximately 1000-fold in vitro. YF1 also controls gene expression in a light-dependent manner in vivo. Signals are transmitted from the light-oxygen-voltage sensor domain to the histidine kinase domain via a 40 degrees -60 degrees rotational movement within an alpha-helical coiled-coil linker; light is acting as a rotary switch. These signaling principles are broadly applicable to domains linked by alpha-helices and to chemo- and photosensors. Conserved sequence motifs guide the rational design of light-regulated variants of histidine kinases and other proteins.

Signal transduction proteins are organized into sensor (input) domains that perceive a signal and, in response, regulate the biological activity of effector (output) domains. We reprogrammed the input signal specificity of a normally oxygen-sensitive, light-inert histidine kinase by replacing its chemosensor domain by a light-oxygen-voltage photosensor domain. Illumination of the resultant fusion kinase YF1 reduced net kinase activity by approximately 1000-fold in vitro. YF1 also controls gene expression in a light-dependent manner in vivo. Signals are transmitted from the light-oxygen-voltage sensor domain to the histidine kinase domain via a 40 degrees -60 degrees rotational movement within an alpha-helical coiled-coil linker; light is acting as a rotary switch. These signaling principles are broadly applicable to domains linked by alpha-helices and to chemo- and photosensors. Conserved sequence motifs guide the rational design of light-regulated variants of histidine kinases and other proteins.

FixL from Bradyrhizobium japonicum is a PAS sensor protein in which two PAS domains covalently linked to a histidine kinase domain are responsible for regulating nitrogen fixation in an oxygen-dependent manner. The more C-terminal PAS domain, denoted bjFixLH, contains a heme cofactor that binds diatomic molecules such as carbon monoxide and oxygen and regulates the activity of the FixL histidine kinase as part of a two-component signaling system. We present the structures of ferric, deoxy, and carbon monoxide-bound bjFixLH in a new space group ( P1) and at resolutions (1.5-1.8 A) higher than the resolutions of those previously obtained. Interestingly, bjFixLH can form two different dimers (in P1 and R32 crystal forms) in the same crystallization solution, where the monomers in one dimer are rotated approximately 175 degrees relative to the second. This suggests that PAS monomers are plastic and that two quite distinct quaternary structures are closely similar in free energy. We use screw rotation analysis to carry out a quantitative pairwise comparison of PAS quaternary structures, which identifies five different relative orientations adopted by isolated PAS monomers. We conclude that PAS monomer arrangement is context-dependent and could differ depending on whether the PAS domains are isolated or are part of a full-length protein. Structurally homologous residues comprise a conserved dimer interface. Using network analysis, we find that the architecture of the PAS dimer interface is continuous rather than modular; the network of residues comprising the interface is strongly connected. A continuous dimer interface is consistent with the low dimer-monomer dissociation equilibrium constant. Finally, we quantitate quaternary structural changes induced by carbon monoxide binding to a bjFixLH dimer, in which monomers rotate by up to approximately 2 degrees relative to each other. We relate these changes to those in other dimeric PAS domains and discuss the role of quaternary structural changes in the signaling mechanisms of PAS sensor proteins.

An understanding of how allostery, the conformational coupling of distant functional sites, arises in highly evolvable systems is of considerable interest in areas ranging from cell biology to protein design and signaling networks. We reasoned that the rigidity and defined geometry of an α-helical domain linker would make it effective as a conduit for allosteric signals. To test this idea, we rationally designed 12 fusions between the naturally photoactive LOV2 domain from Avena sativa phototropin 1 and the Escherichia coli trp repressor. When illuminated, one of the fusions selectively binds operator DNA and protects it from nuclease digestion. The ready success of our rational design strategy suggests that the helical “allosteric lever arm” is a general scheme for coupling the function of two proteins.

We have examined the interaction between olfactory ensheathing cells (OECs), Schwann cells (SC), oligodendrocytes, and CNS axons using cultures generated from embryonic rat spinal cord. Oligodendrocyte process extension and myelination in these cultures was poor if the cells were plated on OECs or SCs. Myelin internodes and nodes of Ranvier formed frequently if these cultures were plated onto monolayers of neurosphere-derived astrocytes (NsAs). In the myelinated fibers generated on NsAs, Nav channels, caspr, and neurofascin molecules were correctly assembled at the nodes of Ranvier. The density of neurites, survival, and antigenic differentiation of oligodendrocytes was similar on OEC and NsAs monolayers. However, on OEC monolayers, despite a transient increase in the number of endogenous oligodendrocytes, there was a decrease in oligodendrocyte process extension and axonal ensheathment when compared with cultures plated on NsAs monolayers. To determine if these changes were due to axonal or glial factors, spinal cord oligodendrocytes were plated onto monolayers of OECs, NsAs, and poly-L-lysine in the absence of neurons. In these cultures, process extension and myelin-like membrane formation by oligodendrocytes was improved on monolayers of OEC. This suggests that inhibition of process extension is mediated via cross-talk between OECs and neurites. In cultures containing axons plated on OEC monolayers, oligodendrocyte process formation, axonal ensheathment, and myelination occurred albeit lower if the cultures were supplemented with NsAs conditioned medium. These data suggest OECs can permit neurite extension and oligodendrocyte proliferation, but lack secreted factor(s) and possible cell-cell contact that is necessary for oligodendrocyte process extension and myelination.

The photosensor YtvA binds flavin mononucleotide and regulates the general stress reaction in Bacillus subtilis in response to blue light illumination. It belongs to the family of light-oxygen-voltage (LOV) proteins that were first described in plant phototropins and form a subgroup of the Per-Arnt-Sim (PAS) superfamily. Here, we report the three-dimensional structure of the LOV domain of YtvA in its dark and light states. The protein assumes the global fold common to all PAS domains and dimerizes via a hydrophobic interface. Directly C-terminal to the core of the LOV domain, an alpha-helix extends into the solvent. Light absorption causes formation of a covalent bond between a conserved cysteine residue and atom C(4a) of the FMN ring, which triggers rearrangements throughout the LOV domain. Concomitantly, in the dark and light structures, the two subunits of the dimeric protein rotate relative to each other by 5 degrees . This small quaternary structural change is presumably a component of the mechanism by which the activity of YtvA is regulated in response to light. In terms of both structure and signaling mechanism, YtvA differs from plant phototropins and more closely resembles prokaryotic heme-binding PAS domains.

NifL is a multidomain sensor protein responsible for the transcriptional regulation of genes involved in response to changes in cellular redox state and ADP concentration. Cellular redox is monitored by the N-terminal PAS domain of NifL which contains an FAD cofactor. Flavin-based PAS domains of this type have also been referred to as LOV domains. To explore the mechanism of signal recognition and transduction in NifL, we determined the crystal structure of the FAD-bound PAS domain of NifL from Azotobacter vinelandii to 1.04 A resolution. The structure reveals a novel cavity within the PAS domain which contains two water molecules directly coordinated to the FAD. This cavity is connected to solvent by multiple access channels which may facilitate the oxidation of the FAD by molecular oxygen and the release of hydrogen peroxide. The structure contains a dimer of the NifL PAS domain that is structurally very similar to those described in other crystal structures of PAS domains and identifies a conserved dimerization motif. An N-terminal amphipathic helix constitutes part of the dimerization interface, and similar N-terminal helices are identified in other PAS domain proteins. The structure suggests a model for redox-mediated signaling in which a conformational change is initiated by redox-dependent changes in protonation at the N5 atom of FAD that lead to reorganization of hydrogen bonds within the flavin binding pocket. A structural signal is subsequently transmitted to the beta-sheet interface between the monomers of the PAS domain.

The Escherichia coli protein YcgF contains a photosensory flavin adenine dinucleotide (FAD)-binding BLUF domain covalently linked to an EAL domain, which is predicted to have cyclic-di-guanosine monophosphate (GMP) phosphodiesterase activity. We have cloned, overexpressed and purified this protein, which we refer to as blue light-regulated phosphodiesterase (Blrp) for its putative activity. Blrp undergoes a reversible photocycle after exposure to light in which the spectrum of its photostationary state and kinetics of recovery of the dark state are similar to those of the isolated BLUF domain of the AppA protein. Unlike the AppA BLUF domain, the chromophore environment in the context of full-length Blrp is asymmetric, and the protein does not undergo any detectable global changes on exposure to blue light. When overexpressed in E. coli, Blrp copurifies with certain proteins which suggests that it plays a protective role in response to oxidative stress. Predicted proteins from Klebsiella pneumoniae and from a bacterium in the Sargasso Sea are similar to E. coli Blrp in both their BLUF and EAL domains, which suggests that blue light sensing in these bacteria may follow similar pathways.

The phototropins are flavoprotein kinases that control phototropic bending, light-induced chloroplast movement, and stomatal opening in plants. Two flavin mononucleotide binding light, oxygen, or voltage (LOV) domains are the sites for initial photochemistry in these blue light photoreceptors. We have determined the steady state, photoexcited crystal structure of a flavin-bound LOV domain. The structure reveals a unique photochemical switch in the flavin binding pocket in which the absorption of light drives the formation of a reversible covalent bond between a highly conserved Cys residue and the flavin cofactor. This provides a molecular picture of a cysteinyl-flavin covalent adduct, the presumed signaling species that leads to phototropin kinase activation and subsequent signal transduction. We identify closely related LOV domains in two eubacterial proteins that suggests the light-induced conformational change evident in this structure is an ancient biomolecular response to light, arising before the appearance of plants.

Light, oxygen, or voltage (LOV) domains constitute a new class of photoreceptor proteins that are sensitive to blue light through a noncovalently bound flavin chromophore. Blue-light absorption by the LOV2 domain initiates a photochemical reaction that results in formation of a long-lived covalent adduct between a cysteine and the flavin cofactor. We have applied ultrafast spectroscopy on the photoaccumulated covalent adduct state of LOV2 and find that, upon absorption of a near-UV photon by the adduct state, the covalent bond between the flavin and the cysteine is broken and the blue-light-sensitive ground state is regained on an ultrafast time scale of 100 ps. We thus demonstrate that the LOV2 domain is a reversible photochromic switch, which can be activated by blue light and deactivated by near-UV light. Copyright © 2004 American Chemical Society.

The modular nature of proteins containing PAS (Per-ARNT-Sim) or other sensor domains enables signaling networks to be diverse and poses an interesting question: how can sensor domains with largely conserved tertiary structures regulate effector domains with very diverse structures and functions? We address this question by examining signal processing by the PAS sensor domain, which can regulate the activity of covalently linked effector domains such as a kinase, phosphodiesterase or DNA binding domains. In many cases oligomerization of sensor proteins is essential for signal transduction. We present the structure of a heme-PAS domain dimer from Bradyrhizobium japonicum (bjFixLH) in a new space group (P1) and at higher resolutions (1.5-1.8 A) than those previously obtained. Interestingly, bjFixLH can form two different dimers in the same crystallization solution, where the monomers in one dimer are rotated ∼175° relative to the second. This suggests that PAS monomers are plastic and that two quite distinct quaternary structures are closely similar in free energy. Comparison of PAS domain dimers using screw rotation analysis reveals that PAS monomers adopt a discrete range of monomer orientations. Similar to the light-sensitive PAS domain YtvA-LOV from Bacillus subtilis, bjFixLH undergoes signal-induced quaternary structural changes where monomers rotate ∼2° relative to each other. Signal-induced quaternary structural changes accommodate the ability of PAS sensor domains to regulate a wide variety of effector domains since PAS and effector domains would not be required to interact with each other in a structure-specific manner. Our results will guide the rational design of novel PAS signaling proteins.

Cytochrome oxidase from Pseudomonas aeruginosa has been crystallized from 2 m -ammonium sulfate. The crystals occur principally as thin diamond-shaped plates of space group P 2 1 2 1 2 with unit cell dimensions of 92 A × 115 A × 76 A. Determination of the density of glutaraldehyde-fixed, water-equilibrated crystals (1.167 g/cm 3 ), coupled with the unit cell volume (804,000 A 3 ), indicates that there is one subunit (~63,000 M r) per asymmetric unit. X-ray diffraction data which were limited to 12 A resolution due to small crystal size were obtained for the hk 0 and 0 kl zones using precession photography. Amplitude and phase data for the hk 0, 0 kl , and h 0 l zones were obtained from computer-based Fourier analysis of appropriate micrographs recorded from negatively stained microplates and thin sections of larger crystals using minimal beam electron microscopy. For crystals embedded in the presence of tannic acid it was possible to achieve 20 A resolution which is comparable to the resolution achieved with negative staining of thin crystalline arrays. In addition, unstained electron diffraction on glutaraldehyde-fixed, glucose-stabilized plates was recorded to a resolution of 9 A. The three-dimensional packing of the cytochrome oxidase dimer in the unit cell has been deduced from computer reconstructed images of the three principal projections along the crystallographic axes. The cytochrome oxidase dimer is located in the unit cell with the dimer axis coincident with a crystallographic 2-fold axis; thus within the resolution of the present data in projection (9 A) the two subunits are identical, in agreement with biochemical evidence. The crystals have been prepared with the enzyme in the fully oxidized state and upon reduction a progressive cracking of the crystals is observed, possibly due to a conformational change dependent on the oxidation state of the heme iron.

DesHis (146β) hemoglobin is a β chain modification of human hemoglobin in which the COOH-terminal histidine 146β has been removed by digestion with carboxypeptidase B. Previous crystallographic investigations of the structures of desHis deoxyhemoglobin and deoxyhemoglobin Hiroshima (His 146β → Asp) suggest that the atomic structures of these derivatives differ from each other and from hemoglobin A only in the immediate environment of the COOH-terminal residue. Kinetic studies of the binding of oxygen, carbon monoxide, and n-butyl isocyanide to desHis hemoglobin reveal that the properties of desHis hemoglobin and hemoglobin Hiroshima are indeed similar, but not identical; the properties of both differ appreciably from those of hemoglobin A. These qualitative differences in kinetic properties are not readily reconciled with the apparently minor structural differences revealed in the structural studies. They suggest rather that the unliganded forms of both derivatives adopt a new conformation, or series of conformations in slow equilibrium, which are distinct from the conformations of either deoxy- or oxyhemoglobin A.

Background: Restrictocin, a highly specific ribotoxin made by the fungus Aspergillus restrictus , cleaves a single phosphodiester bond in the 28S RNA of eukaryotic ribosomes, inhibiting protein synthesis. The sequence around this cleavage site is a binding site for elongation factors, and is conserved in all cytoplasmic ribosomes. The catalytic mechanism of restrictocin and the reasons for its high substrate specificity are unknown. No structure has been determined for any other member of the Aspergillus ribotoxin family. Results The crystal structure of restrictocin was determined at 2.1 a resolution by single isomorphous replacement and anomalous scattering techniques, and refined to 1.7 a resolution using synchrotron Laue data. The structural core of the protein, in which a three-turn α helix is packed against a five-stranded antiparallel β sheet, can be well aligned with that of ribonuclease T1. Large positively charged peripheral loops near the active site construct a platform with a concave surface for RNA binding. Conclusion Restrictocin appears to combine the catalytic components of T1 ribonucleases with the base recognition components of Sa ribonucleases. Modeling studies using an NMR structure of an RNA substrate analog suggest that the tertiary structure of the substrate RNA is important in protein–RNA recognition, fitting closely into the concavity of the presumed binding site. We speculate that the large 39-residue loop L3, which has similarities to loops found in lectin sugar-binding domains, may be responsible for restrictocin's ability to cross cell membranes.

Phytochromes are red-light photoreceptors that were first characterized in plants, with homologs in photosynthetic and non-photosynthetic bacteria known as bacteriophytochromes (BphPs). Upon absorption of light, BphPs interconvert between two states denoted Pr and Pfr with distinct absorption spectra in the red and far-red. They have recently been engineered as enzymatic photoswitches for fluorescent-marker applications in non-invasive tissue imaging of mammals. This article presents cryo- and room-temperature crystal structures of the unusual phytochrome from the non-photosynthetic myxobacterium Stigmatella aurantiaca (SaBphP1) and reveals its role in the fruiting-body formation of this photomorphogenic bacterium. SaBphP1 lacks a conserved histidine (His) in the chromophore-binding domain that stabilizes the Pr state in the classical BphPs. Instead it contains a threonine (Thr), a feature that is restricted to several myxobacterial phytochromes and is not evolutionarily understood. SaBphP1 structures of the chromophore binding domain (CBD) and the complete photosensory core module (PCM) in wild-type and Thr-to-His mutant forms reveal details of the molecular mechanism of the Pr/Pfr transition associated with the physiological response of this myxobacterium to red light. Specifically, key structural differences in the CBD and PCM between the wild-type and the Thr-to-His mutant involve essential chromophore contacts with proximal amino acids, and point to how the photosignal is transduced through the rest of the protein, impacting the essential enzymatic activity in the photomorphogenic response of this myxobacterium.

The structure of vitamin D-dependent calcium-binding protein from bovine intestine, determined crystallographically to 2.3 A, contains a pair of calcium-binding domains similar to those in parvalbumin, but one domain has a longer and rearranged calcium-binding loop. This result has implications for structure predictions of other calcium-binding proteins such as calmodulin and S-100.

Publisher Summary An important step in understanding the operation of regulatory calcium-binding proteins such as calmodulin (CAM) is determination of their three-dimensional structures with and without calcium bound. X-Ray crystallography is the method of choice for such determinations, provided that the proteins to be studied can be crystallized. Structures are determined, which reveal the conformation of three fully calcium-loaded proteins in the CaM family, and a fourth related protein, troponin C (Tn-C), is crystallized in a half-saturated form; its structure contains two calcium-loaded binding domains and two calcium-free domains. The determination of the structure of the vitamin D-dependent protein from bovine intestine, ICaBP is also described. Structural details to be examined in a protein molecule include secondary structure such as a helices and 3 sheet-binding sites such as the calcium-binding sites in ICaBP, hydrogen bonds, salt bridges, and hydrophobic contacts. Helices and sheets are generally apparent by examination of a model or stereo image of the backbone of the molecule. Secondary structure in ICaBP is explored and illustrated graphically in this chapter.

Publisher Summary This chapter elaborates some of the ways in which EF-hand proteins have diversified and to speculate concerning possible variations yet to be discovered on the EF-hand theme. Calcium- binding sites in proteins coordinate the calcium ion by protein oxygen atoms and water molecules in a roughly octahedral arrangement. The prototypic EF-hand presumably gained calcium-binding ability in a similar way by modification of a protein or a protein fragment, which originally had some other function. The archetypal EF-hand protein is calmodulin, a four-domain protein whose two halves are similar but not identical. Parvalbumin (Pa) has only three domains, one of which has lost the ability to bind calcium. The 9 Kilodalton vitamin D-dependent calcium-binding protein or calbindin (CaBP ) consists of just two domains, one of which is a variant EF-hand in which the majority of the calcium ligands are main chain carbonyls. Some myosin light chains, although retaining a four-domain structure, have entirely lost the ability to bind calcium and have presumably acquired some other function in muscle contraction. There are now a number of EF-hand proteins for which amino acid sequences but no three-dimensional structure are available.

Phytochrome proteins control the growth, reproduction, and photosynthesis of plants, fungi, and bacteria. Light is detected by a bilin cofactor, but it remains elusive how this leads to activation of the protein through structural changes. We present serial femtosecond X-ray crystallographic data of the chromophore-binding domains of a bacterial phytochrome at delay times of 1 ps and 10 ps after photoexcitation. The data reveal a twist of the D-ring, which leads to partial detachment of the chromophore from the protein. Unexpectedly, the conserved so-called pyrrole water is photodissociated from the chromophore, concomitant with movement of the A-ring and a key signaling aspartate. The changes are wired together by ultrafast backbone and water movements around the chromophore, channeling them into signal transduction towards the output domains. We suggest that the observed collective changes are important for the phytochrome photoresponse, explaining the earliest steps of how plants, fungi and bacteria sense red light.