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Bioluminescence is the phenomenon of light emission from an organism and occurs as a result of a reaction between a luciferase and a luciferin. One of the most widespread luciferins is coelenterazine, which demonstrate a considerable presence in marine food chains due to its biosynthesis in low-tier species such as decapod shrimp, ctenophores, and copepods. Coelenterazine is synthesized from L-phenylalanine and L-tyrosine however, the biosynthetic precursor to the luciferin remains unknown. Notably, a study of the ctenophore Mnemiopsis leidyi, which has demonstrated biosynthesis of coelenterazine, identified an isopenicillin-N-synthase homolog as a possible precursor to the substrate. Here, a suggested biosynthetic pathway for coelenterazine is explored, involving an isopenicillin-N-synthase homolog with a C-terminal Phe-Tyr-Tyr-motif. The homolog-protein is studied through an in vitro subject, in addition to the ctenophore Mnemiopsis leidyi. The experimental approach involves a previously established expression and purification protocol, peptide mapping, fluorescence measurements, crystallization, and proteome analysis. In this project, evidence is provided for the biosynthetic relation of FYY-protein as a precursor to coelenterazine., the results identify the ctenophore O. maculata as a possible coelenterazine producer. Additionally, a proteome of non-starved M. leidyi comprising 3675 gene products, 479 gene products with no known homolog. In addition, increased yield for in vitro studies may be achieved by revising the current purification protocol to facilitate protein incubation with iron(II). Noting that ultimately, the primary cause of insufficient yield of homogenous sample is the analytic distribution of the expressed FYY2-protein.

A genetic selection system for activity of HIV protease is described that is based on a synthetic substrate constructed as a modified AraC regulatory protein that when cleaved stimulate L-arabinose metabolism in an Escherichia coli araC strain. Growth stimulation on selective plates was shown to depend on active HIV protease and the scissile bond in the substrate. In addition, the growth of cells correlated well with the established cleavage efficiency of the sites in the viral polyprotein, Gag, when these sites were individually introduced into the synthetic substate of the selection system. Plasmids encoding protease variants selected based on stimulation of cell growth in the presence of saquinavir or cleavage of a site not cleaved by wild-type protease, were indistinguishable with respect to both phenotypes. Also, both groups of selected plasmids encoded side chain substitutions known from clinical isolates or displayed different side chain substitutions but at identical positions. One highly frequent side chain substitution, E34V, not regarded as a major drug resistance substitution was found in variants obtained under both selective conditions and is suggested to improve protease processing of the synthetic substrate. This substitution is away from the substrate-binding cavity and together with other substitutions in the selected reading frames supports the previous suggestion of a substrate-binding site extended from the active site binding pocket itself.

Electrostatic forces are important for protein folding and are favored targets of protein engineering. However, interactions between charged residues are difficult to study because of the complex network of interactions found in most proteins. We have designed a purposely simple system to investigate this problem by systematically introducing individual and pairs of charged and titratable residues in a protein otherwise free of such residues. We used constant pH molecular dynamics simulations, NMR spectroscopy, and thermodynamic double mutant cycles to probe the structure and energetics of the interaction between the charged residues. We found that the partial burial of surface charges contributes to a shift in pKa value, causing an aspartate to titrate in the neutral pH range. Additionally, the interaction between pairs of residues was found to be highly context dependent, with some pairs having no apparent preferential interaction, while other pairs would engage in coupled titration forming a highly stabilized salt bridge. We find good agreement between experiments and simulations and use the simulations to rationalize our observations and to provide a detailed mechanistic understanding of the electrostatic interactions.

Computational protein design has taken big strides in recent years; however, the tools available are still not at a state where a sequence can be designed to fold into a given protein structure at will and with high probability. We have applied here a recent release of Rosetta Design to redesign a set of structurally very similar proteins belonging to the thioredoxin fold. We used a genetic screening tool to estimate solubility/folding of the designed proteins in E. coli and to select the best hits from this for further biochemical characterization. We have previously used this set of template proteins for redesign and found that success was highly dependent on template structure, a trait which was also found in this study. Nevertheless, state-of-the-art design software is now able to predict the best template, most likely due to the introduction of an energy term that reports on stress in covalent bond lengths and angles. The template that led to the greatest fraction of successful designs was the same (a thioredoxin from spinach) as that identified in our previous study. Our previously described redesign of thioredoxin, which also used the spinach protein as a template, however also performed well as a template. In the present study, both of these templates yielded proteins with compact folded structures and enforced the conclusion that any design project must carefully consider different design templates. Fortunately, selecting designs based on energies appears to correctly identify such templates.

Does sodium dodecyl sulfate (SDS) denature proteins through electrostatic SDS-protein interactions? We show that a protein completely lacking charged side chains is unfolded by SDS in a manner similar to charged proteins, revealing that formal protein charges are not required for SDS-induced protein unfolding or binding.

Gaussia luciferase is a poorly characterized luciferase, in a poorly characterized field. The arbitrary words "flash" and "glow" kinetics are often used to characterize the reaction between luciferase and luciferin, both terms are however imprecise and does not adequately describe the bioluminescent reaction. Several approaches of characterization was pursued, including new mutant constructs, NMR and Bioluminescence assays. While some of these approaches was never finished due to unforeseen events, GLuc was discovered to exhibit some form of product inhibition and deactivation, in addition to illumination of other aspects of this protein. The exact mechanism of GLuc is still not known. however two hypothetical models have been theorized and discussed, in an attempt to elucidate the true meaning of the words flash and glow kinetics.

M2 proton kanalen er et transmembran protein der sidder i influenza A virusen, og er ansvarlig for proton flux ind i virusen når endosomet i vært cellen sænker pH. Det er et vigtigt trin i viral replikation. M2 kanalen kan inhiberes af to kommercielt solgte stoffer, Amantadine og Rimantadine. De fleste nuværende influenza vira er dog blevet resistente overfor disse inhibitorer, hovedsaglig igennem en enkelt mutation i det transmembrane domæne af M2. Et fluorescens baseret proton konduktions (pHlux) assay blev udvilket til at undersøge M2 varianter og deres inhibering. I denne opgave blev dette assay brugt til at undersøge tre nye mutationer ved kendte resistens positioner i M2 kanalen. De blev undersøgt både for deres funktion, og for bindingen til Amantadine, Rimantadine, og tre nye potentielle inhibitorer. Hvor G34A variant virkede resitent var G34S varianten påvirket delvist af alle fem molekyler. L38Q varianten forstyrrede M2 funktionen, hvilket også blev set i en anden L38 mutation. Da der er nærmest total resistens i nulevende influenza vira, kan information omkring hvordan mutationer påvirker M2 kanalens binding til inhibitorer forhåbentligt hjælpe til at finde nye, stærke M2 inhibitorer., The M2 proton channel is a transmembrane protein in the viral envelope of the influenza A virus responsible for the influx of protons into the virion that occurs after host cell endosomal acidification. This is a crucial step in viral replication. The M2 channel has been the target of two commercially available drugs, whose active compounds are Amantadine and Rimantadine. However, most current influenza strains are resistant to these two inhibitors, mainly through a single mutation in the transmembrane domain of M2. A fluorescence based proton conduction (pHlux) assay was developed to examine M2 proton channel variants and their inhibition by drugs. In this study, the assay is used to examine three new single point mutation variants in the M2 channel at known resistance positions. They were examined both for their channel function and their binding to Amantadine, Rimantadine, and three new potential inhibitors. While the G34Avariantseemedresistant,theG34SvariantofM2appearedtobesomewhat sensitive to all five inhibitors. The L38Q variant disrupted normal M2 function, aneffectpossiblyseeninanotherL38mutation.Withnearcompleteresistancein current influenza strains in circulation, increased information about how mutations in the M2 channel affect binding to inhibitors can hopefully aid in the design of stronger drugs.

It was intended for GLuc-C9D, a variant of the Gaussia Luciferase with a C-term Solubility Enhancing Peptide (SEP)-Tag to be expressed in E. Coli for structural characterization. Through SDS-PAGE under non-reducing conditions it was observed that GLuc-C9D aggregates into disulphide-linked polymers while staying in solution when expressed in standard BL21(DE3)-cells. Expression of GLuc-C9D as a monomeric protein was increased by co-expression of the sulfhydryl oxidase, Erv1p, and a human Protein Disulphide Isomerase. It was possible to separate monomeric from polymeric GLuc-C9D by gel-filtration and ammonium sulphate precipitation. Data obtained by Circular Dichroism indicated a mostly unstructured protein with ~20 % helical and sheet formation. At the time of writing, it was discovered that the gene for GLuc-TG, a E100A+G103 variant, had been supplied rather than GLuc-C9D. The findings are expected to remain unaffected by this discovery.

A circularly permutated variant of Green Fluorescent Protein (GFP), with strand 10 in the N- terminal of the protein, was successfully expressed and purified. Trypsin digest, followed by size exclusion chromatography under denaturing conditions lead to the successful removal of strand 10 from the remainder of GFP (truncated GFP). Upon complementation of truncated GFP with synthetic strand 10, the complex regained fluorescence, best explained by a double exponential association fit. The kinetics and fluorescence were pH dependent, but not affected significantly by up to 500 mM NaCl. A strand 10 that did not lead to fluorescence upon complementation was shown to still bind, and affect the kinetics upon adding a strand 10 that lead to fluorescence. Complementation was shown to be dependent on strand length, with three different lengths tested. The shortest strand exhibited the fastest kinetics. Based on the studies, a binding mechanism containing two parallel pathways was suggested.

As part of a larger project focusing on computational protein design, a large number of sequences had been designed, aiming to achieve a thioredoxin-like fold. In order to provide feedback for the design process, a selection system was proposed for in vivo testing of the designs. The system is based on fusion of the designed thioredoxins with a selective partner, OPRTase, such that the folding of the designs would affect the activity of the OPRTase. This system was constructed and used to test a selection of the designs, via assays screening for activity and temperature sensitivity. In addition, it was attempted to use the system as a selection tool for improving the designs, both via selecting for spontaneous mutants and using UV mutagenesis. The system was overall quite effective and appears to give useful information about the designs. The results achieved in vivo are largely compatible with the behavior of the designs in vitro. However, the final selection was not successful in producing improvements in the designs.

The superfamily of G protein-coupled receptors plays a major role in conducting signals across the cell membrane and offers enormous scope for new therapeutic targets. Predicting receptor-ligand interactions based on available structural information forms the physical basis for drug discovery in the pharmaceutical industry. Among all GPCRs, members of the ghrelin receptor family have been shown to be involved in a diverse set of medical conditions, such as type-2 diabetes, obesity, Parkinson's disease and schizophrenia. The recently solved x-ray crystal structure of the agonist- bound neurotensin receptor 1 provides a unique opportunity for the exploration of the structural basis of ligand binding to other closely related members of the ghrelin receptor family. This study investigates the underlying mechanism of small molecule binding to three receptors within the ghrelin receptor family: the ghrelin receptor (GHSR) and the two neurotensin receptor subtypes (NTSR1 and NTSR2). A combined experimental and computational approach was made to systematically probe receptor interactions with a diverse set of small molecules showing distinct pharmacological profiles. In vitro mutational mapping based on inositol-phosphate accumulation assays indicated potential residues in the binding site of GHSR that are involved in binding of the small molecule compounds Bayer YIL781 and Abbott 14c. Computational modeling and docking studies yielded novel insights into structural features of the ghrelin receptor. Most importantly, a potential salt-bridge interaction between TM-III and TM-VI was proposed that suggests reinterpretation of previous studies. Docking studies in the two neurotensin receptor subtypes, NTSR1 and NTSR2, indicated a striking overlap between the binding modes of the small molecule compounds SR48692, SR142948A and the endogenous agonist neurotensin, despite their overall distinct pharmacological effects. Taken together, these results revealed structural feat

We present the application of a redox-sensitive mutant of the yellow fluorescent protein (rxYFP) to image, with elevated sensitivity and high temporal and spatial resolution, oxidative responses of eukaryotic cells to pathophysiological stimuli. The method presented, based on the ratiometric quantitation of the distribution of fluorescence by confocal microscopy, allows us to draw real-time "redox maps" of adherent cells and to score subtle changes in the intracellular redox state, such as those induced by overexpression of redox-active proteins. This strategy for in vivo imaging of redox signaling circumvents many of the technical limitations currently encountered in the study of complex redox-based phenomena and promises to contribute substantially to this expanding area of signal transduction.