After more than one-half century of investigations, the mechanism of bioluminescence from the FMNH2 assisted oxygen oxidation of an aliphatic aldehyde on bacterial luciferase continues to resist elucidation. There are many types of luciferase from species of bioluminescent bacteria originating from both marine and terrestrial habitats. The luciferases all have close sequence homology, and in vitro, a highly efficient light generation is obtained from these natural metabolites as substrates. Sufficient exothermicity equivalent to the energy of a blue photon is available in the chemical oxidation of the aldehyde to the corresponding carboxylic acid, and a luciferase-bound FMNH-OOH is a key player. A high energy species, the source of the exothermicity, is unknown except that it is not a luciferin cyclic peroxide, a dioxetanone, as identified in the pathway of the firefly and the marine bioluminescence systems. Besides these natural substrates, variable bioluminescence properties are found using other reactants such as flavin analogs or aldehydes, but results also depend on the luciferase type. Some rationalization of the mechanism has resulted from spatial structure determination, NMR of intermediates and dynamic optical spectroscopy. The overall light path appears to fall into the sensitized class of chemiluminescence mechanism, distinct from the dioxetanone types.

Bioluminescence is a chemiluminescence in which one of the participants is a protein and where the light emission serves a functional purpose for the organism in which the protein is found. Bioluminescence with bacterial luciferase, henceforth just referred to as luciferase, is obtained in a reaction with FMNH2 and a long-chain aliphatic aldehyde such as tetradecanal in an air-saturated solution. The rationale for many of the studies of bacterial bioluminescence as well as other bioluminescence systems is derived from the known mechanisms and concepts that have evolved in studies of chemiluminescence. An important step in the understanding of the mechanism of the bioluminescence would be the identification of the chemical structure of the fluorophore responsible for the bioluminescence from the reaction of luciferase alone. For a biophysical description of the bioluminescence mechanism, the first question to be posed is as to the identity of the light-emitting molecule.

Author(s): Lee, Donghyun John | Advisor(s): Whaley, Katharine B | Abstract: This thesis is concerned with tractable methods for modeling the optical and electronic properties of photosynthetic light harvesting complexes. We begin by investigating the validity of the commonly used Frenkel Exciton Hamiltonian, and find that it is inaccurate at the intermolecular distances commonly encountered in photosynthetic systems. We introduce semi-empirical tight-binding Hamiltonians and dynamics models that are better able to simulate the electronic properties of these chromophores at the proper distances and orientations. These models are validated by comparisons of the predictions to experimental results.

The molecular mechanisms of the bioluminescence systems of the firefly, bacteria and those utilizing imidazopyrazinone luciferins such as coelenterazine are gradually being uncovered using modern biophysical methods such as dynamic (ns-ps) fluorescence spectroscopy, NMR, X-ray crystallography and computational chemistry. The chemical structures of all reactants are well defined, and the spatial structures of the luciferases are providing important insight into interactions within the active cavity. It is generally accepted that the firefly and coelenterazine systems, although proceeding by different chemistries, both generate a dioxetanone high-energy species that undergoes decarboxylation to form directly the product in its S-1 state, the bioluminescence emitter. More work is still needed to establish the structure of the products completely. In spite of the bacterial system receiving the most research attention, the chemical pathway for excitation remains mysterious except that it is clearly not by a decarboxylation. Both the coelenterazine and bacterial systems have in common of being able to employ antenna proteins, lumazine protein and the green-fluorescent protein, for tuning the color of the bioluminescence. Spatial structure information has been most valuable in informing the mechanism of the Ca2+-regulated photoproteins and the antenna protein interactions.

We investigated the foaming mechanisms associated with the gas counter pressure (GCP) and mold-opening processes during foam injection molding for products with high quality surface. The molding process involved injecting a polymer/nitrogen solution into a nitrogen-pressurized mold cavity. The preloaded cavity pressure (that is, the GCP) prevented cell nucleation and growth within the polymer melt. This was the case until the GCP was released (without opening the mold) or until the melt filled the mold cavity completely. Then the cavity thickness was suddenly increased by opening the mold. In an attempt to understand the mechanism of cell nucleation and growth, we monitored the pressure profile at several locations in the mold cavity during the molding process. We found that the pressure history in the mold cavity, both as a function of space and time, was the dominant factor that determined cell morphology. By applying the GCP and the mold-opening process, the cell size uniformity was improved when compared with conventional foam injection molding. These methods did not compromise the achievable void fraction. Using GCP and the mold-opening process also significantly improved the surface qualities of the foamed parts. This was because the premature cell growth at the flow front during the mold filling stage had been eliminated.

The lux-operon of bioluminescent bacteria contains the genes coding for the enzymes required for light emission. Some species of Photobacteria feature an additional gene, luxF, which shows similarity to luxA and luxB, the genes encoding the heterodimeric luciferase. Isolated dimeric LuxF binds four molecules of an unusually derivatized flavin, i.e., 6-(3′-(R)-myristyl)-FMN (myrFMN). In the present study we have heterologously expressed LuxF in Escherichia coli BL21 in order to advance our understanding of the protein's binding properties and its role in photobacterial bioluminescence. Structure determination by X-ray crystallography confirmed that apo-LuxF possesses four preorganized binding sites, which are further optimized by adjusting the orientation of amino acid side chains. To investigate the binding properties of recombinant LuxF we have isolated myrFMN from Photobacterium leiognathi S1. We found that LuxF binds myrFMN tightly with a dissociation constant of 80 ± 20 nM demonstrating that the purified apo-form of LuxF is fully competent in myrFMN binding. In contrast to LuxF, binding of myrFMN to luciferase is much weaker (Kd = 4.0 ± 0.4 μM) enabling LuxF to prevent inhibition of the enzyme by scavenging myrFMN. Moreover, we have used apo-LuxF to demonstrate that myrFMN occurs in all Photobacteria tested, irrespective of the presence of luxF indicating that LuxF is not required for myrFMN biosynthesis.

Ca2+‐regulated photoproteins are responsible for the bioluminescence of a variety of marine coelenterates. All hydromedusan photoproteins are a single‐chain polypeptide to which 2‐hydroperoxycoelenterazine is tightly but non‐covalently bound. Bioluminescence results from oxidative decarboxylation of 2‐hydroperoxycoelenterazine, generating protein‐bound coelenteramide in an excited state. The bioluminescence spectral maxima of recombinant photoproteins vary in the range 462–495 nm, despite a high degree of identity of amino acid sequences and spatial structures of these photoproteins. Based on studies of obelin and aequorin mutants with substitution of Phe to Tyr and Tyr to Phe, respectively [Stepanyuk GA et al. (2005) FEBS Lett 579, 1008–1014], it was suggested that the spectral differences may be accounted for by an additional hydrogen bond between the hydroxyl group of a Tyr residue and an oxygen atom of the 6‐(p‐hydroxyphenyl) substituent of coelenterazine. Here, we report the crystal structures of two conformation states of the F88Y obelin mutant that has bioluminescence and product fluorescence spectra resembling those of aequorin. Comparison of spatial structures of the F88Y obelin conformation states with those of wild‐type obelin clearly shows that substitution of Phe to Tyr does not affect the overall structures of either F88Y obelin or its product following Ca2+ discharge, compared to the conformation states of wild‐type obelin. The hydrogen bond network in F88Y obelin being due to the Tyr substitution clearly supports the suggestion that different hydrogen bond patterns near the oxygen of the 6‐(p‐hydroxyphenyl) substituent are the basis for spectral modifications between hydromedusan photoproteins.

Ca(2+)-regulated photoproteins, which are responsible for light emission in a variety of marine coelenterates, are a highly valuable tool for measuring Ca(2+) inside living cells. All of the photoproteins are a single-chain polypeptide to which a 2-hydroperoxycoelenterazine molecule is tightly but noncovalently bound. Bioluminescence results from the oxidative decarboxylation of 2-hydroperoxycoelenterazine, generating protein-bound coelenteramide in an excited state. Here, the crystal structures of the Y138F obelin mutant before and after bioluminescence are reported at 1.72 and 1.30 Å resolution, respectively. The comparison of the spatial structures of the conformational states of Y138F obelin with those of wild-type obelin gives clear evidence that the substitution of Tyr by Phe does not affect the overall structure of both Y138F obelin and its product following Ca(2+) discharge compared with the corresponding conformational states of wild-type obelin. Despite the similarity of the overall structures and internal cavities of Y138F and wild-type obelins, there is a substantial difference: in the cavity of Y138F obelin a water molecule corresponding to W2 in wild-type obelin is not found. However, in Ca(2+)-discharged Y138F obelin this water molecule now appears in the same location. This finding, together with the observed much slower kinetics of Y138F obelin, clearly supports the hypothesis that the function of a water molecule in this location is to catalyze the 2-hydroperoxycoelenterazine decarboxylation reaction by protonation of a dioxetanone anion before its decomposition into the excited-state product. Although obelin differs from other hydromedusan Ca(2+)-regulated photoproteins in some of its properties, they are believed to share a common mechanism.

The bright bioluminescence of ctenophores, found in oceans worldwide, is determined by Ca(2+)-regulated photoproteins, functionally identical to and sharing many properties of hydromedusan photoproteins. In contrast, however, the ctenophore photoproteins are extremely sensitive to UV and visible light over the range of their absorption spectrum. The spatial structure of a novel light-sensitive photoprotein from the ctenophore Beroe abyssicola in its apoform bound with three calcium ions is determined at 2.0Å. We demonstrate that the apoberovin is a slightly asymmetrical compact globular protein formed by two domains with a cavity in the center, which exactly retains the fold architecture characteristic of hydromedusan photoproteins despite their low amino acid sequence identity. However, the structural alignment of these two photoprotein classes clearly shows that despite the high similarity of shape and geometry of their coelenterazine-binding cavities, their interiors differ drastically. The key residues appearing to be crucial for stabilizing the 2-hydroperoxycoelenterazine and for formation of the emitter in hydromedusan photoproteins, are replaced in berovin by amino acid residues having completely different side chain properties. Evidently, these replacements must be responsible for the distinct properties of ctenophore photoproteins such as sensitivity to light or the fact that the formation of active photoprotein from apophotoprotein, coelenterazine, and oxygen is more effective at alkaline pH.

The shikimate dehydrogenase (SDH) family consists of enzymes with diverse roles in secondary metabolism. The two most widespread members of the family, AroE and YdiB, function in amino acid biosynthesis and quinate catabolism, respectively. Here, we have determined the crystal structure of an SDH homolog belonging to the RifI class, a group of enzymes with proposed roles in antibiotic biosynthesis. The structure of RifI2 from Pseudomonas putida exhibits a number of distinctive features, including a substantial C-terminal truncation and an atypical mode of oligomerization. The active site of the enzyme contains substrate- and cofactor-binding motifs that are significantly different from those of any previously characterized member of the SDH family. These features are reflected in the novel kinetic properties of the enzyme. RifI2 exhibits much lower activity using shikimate as a substrate than AroE, and a strong preference for NAD(+) instead of NADP(+) as a cofactor. Moreover, the enzyme has only trace activity using quinate, unlike YdiB. Cocrystallization of RifI2 with NAD(+) provided the opportunity to determine the mode of cofactor selectivity employed by the enzyme. We complemented this analysis by probing the role of a strictly conserved residue in the cofactor-binding domain, Asn193, by site directed mutagenesis. This study presents the first crystal structure and formal kinetic characterization of a new NAD(+)-dependent member of the SDH family.

Green-fluorescent protein (GFP) is the origin of the green bioluminescence color exhibited by several marine hydrozoans and anthozoans. The mechanism is believed to be Förster resonance energy transfer (FRET) within a luciferase-GFP or photoprotein-GFP complex. As the effect is found in vitro at micromolar concentrations, for FRET to occur this complex must have an affinity in the micromolar range. We present here a fluorescence dynamics investigation of the recombinant bioluminescence proteins from the jellyfish Clytia gregaria, the photoprotein clytin in its Ca(2+)-discharged form that is highly fluorescent (λ(max) = 506 nm) and its GFP (cgreGFP; λ(max) = 500 nm). Ca(2+)-discharged clytin shows a predominant fluorescence lifetime of 5.7 ns, which is assigned to the final emitting state of the bioluminescence reaction product, coelenteramide anion, and a fluorescence anisotropy decay or rotational correlation time of 12 ns (20 °C), consistent with tight binding and rotation with the whole protein. A 34 ns correlation time combined with a translational diffusion constant and molecular brightness from fluorescence fluctuation spectroscopy all confirm that cgreGFP is an obligate dimer down to nanomolar concentrations. Within the dimer, the two chromophores have a coupled excited-state transition yielding fluorescence depolarization via FRET with a transfer correlation time of 0.5 ns. The 34 ns time of cgreGFP showed no change upon addition of a 1000-fold excess of Ca(2+)-discharged clytin, indicating no stable complexation below 0.2 mM. It is proposed that any bioluminescence FRET complex with micromolar affinity must be one formed transiently by the cgreGFP dimer with a short-lived (millisecond) intermediate in the clytin reaction pathway.

In this review we summarize the progress made towards understanding the role of protein-protein interactions in the function of various bioluminescence systems of marine organisms, including bacteria, jellyfish and soft corals, with particular focus on methodology used to detect and characterize these interactions. In some bioluminescence systems, protein-protein interactions involve an "accessory protein" whereby a stored substrate is efficiently delivered to the bioluminescent enzyme luciferase. Other types of complexation mediate energy transfer to an "antenna protein" altering the color and quantum yield of a bioluminescence reaction. Spatial structures of the complexes reveal an important role of electrostatic forces in governing the corresponding weak interactions and define the nature of the interaction surfaces. The most reliable structural model is available for the protein-protein complex of the Ca2+-regulated photoprotein clytin and green-fluorescent protein (GFP) from the jellyfish Clytia gregaria, solved by means of Xray crystallography, NMR mapping and molecular docking. This provides an example of the potential strategies in studying the transient complexes involved in bioluminescence. It is emphasized that structural studies such as these can provide valuable insight into the detailed mechanism of bioluminescence.

SessionsC779 derivatives and performing SAD experiments on the MX beamlines at the ESRF in Grenoble.Biophysical and biochemical studies have also been performed in order to dissect the role of RecN and its various domains so as to propose a much more accurate mechanism of DSB recognition.

Shikimate dehydrogenase (SDH) catalyzes the reversible NADPH-dependent reduction of 3-dehydroshikimate to shikimate. This reaction represents the fourth step of the shikimate pathway, the essential route for the biosynthesis of the aromatic amino acids in plants, fungi, bacteria, and apicomplexan parasites. The absence of this pathway in animals makes it an attractive target for herbicides and antimicrobials. At least four functionally distinct enzyme classes, AroE, YdiB, SDH-like (SdhL), and AroE-like1 (Ael1), utilize shikimate as a substrate in vitro and form the SDH family. Crystal structures have been determined for AroE, YdiB, and SdhL. In this study, we have determined the first representative crystal structure of an Ael1 enzyme. We demonstrate that Ael1 shares a similar overall structure with the other members of the SDH family. This high level of structural conservation extends to the active sites of the enzymes. In particular, an ionizable active site lysine and aspartate are present in all SDH homologues. Two distinct biochemical roles have been reported for this Lys-Asp pair: as binding residues in YdiB and as a catalytic dyad in AroE and SdhL. Here, we establish that the residues function as a catalytic dyad in Ael1 and, interestingly, in at least one YdiB homologue. The conservation of three-dimensional fold, active site architecture, and catalytic mechanism among members of the SDH family will facilitate the design of drugs targeting the shikimate pathway.

It has been shown that the coelenterazine analog, coelenterazine-v, is an efficient substrate for a reaction catalyzed by Renilla luciferase. The resulting bioluminescence emission maximum is shifted to a longer wavelength up to 40 nm, which allows the use of some "yellow" Renilla luciferase mutants for in vivo imaging. However, the utility of coelenterazine-v in small-animal imaging has been hampered by its instability in solution and in biological tissues. To overcome this drawback, we ligated coelenterazine-v to Ca(2+)-triggered coelenterazine-binding protein from Renilla muelleri, which apparently functions in the organism for stabilizing and protecting coelenterazine from oxidation. The coelenterazine-v bound within coelenterazine-binding protein has revealed a greater long-term stability at both 4 and 37 °C. In addition, the coelenterazine-binding protein ligated by coelenterazine-v yields twice the total light over free coelenterazine-v as a substrate for the red-shifted R. muelleri luciferase. These findings suggest the possibility for effective application of coelenterazine-v in various in vitro assays.

Förster resonance energy transfer within a protein-protein complex has previously been invoked to explain emission spectral modulation observed in several bioluminescence systems. Here we present a spatial structure of a complex of the Ca(2+)-regulated photoprotein clytin with its green-fluorescent protein (cgGFP) from the jellyfish Clytia gregaria, and show that it accounts for the bioluminescence properties of this system in vitro. We adopted an indirect approach of combining x-ray crystallography determined structures of the separate proteins, NMR spectroscopy, computational docking, and mutagenesis. Heteronuclear NMR spectroscopy using variously (15)N,(13)C,(2)H-enriched proteins enabled assignment of backbone resonances of more than 94% of the residues of both proteins. In a mixture of the two proteins at millimolar concentrations, complexation was inferred from perturbations of certain (1)H-(15)N HSQC-resonances, which could be mapped to those residues involved at the interaction site. A docking computation using HADDOCK was employed constrained by the sites of interaction, to deduce an overall spatial structure of the complex. Contacts within the clytin-cgGFP complex and electrostatic complementarity of interaction surfaces argued for a weak protein-protein complex. A weak affinity was also observed by isothermal titration calorimetry (K(D) = 0.9 mM). Mutation of clytin residues located at the interaction site reduced the degree of protein-protein association concomitant with a loss of effectiveness of cgGFP in color-shifting the bioluminescence. It is suggested that this clytin-cgGFP structure corresponds to the transient complex previously postulated to account for the energy transfer effect of GFP in the bioluminescence of aequorin or Renilla luciferase.