Academic literature on the topic 'Chromoproteins'

Proteins of the GFP (green fluorescent protein) family demonstrate a great spectral and phylogenetic diversity. However, there is still an intense demand for red-shifted GFP-like proteins in both basic and applied science. To obtain GFP-like chromoproteins with red-shifted absorption, we performed a broad search in blue-coloured Anthozoa species. We revealed specimens of Actinia equina (beadlet anemone) exhibiting a bright blue circle band at the edge of the basal disc. A novel blue chromoprotein, aeCP597, with an absorption maximum at 597 nm determining the coloration of the anemone basal disk was cloned. AeCP597 carries a chromophore chemically identical with that of the well-studied DsRed (red fluorescent protein from Discosoma sp.). Thus a strong 42-nm bathochromic shift of aeCP597 absorption compared with DsRed is determined by peculiarities of chromophore environment. Site-directed and random mutagenesis of aeCP597 resulted in far-red fluorescent mutants with emission maxima at up to 663 nm. The most bright and stable mutant AQ143 possessed excitation and emission maxima at 595 and 655 nm respectively. Thus aeCP597 and its fluorescent mutants set a new record of red-shifted absorption and emission maxima among GFP-like proteins.

Background: Marine sessile organisms display a color palette that is the result of the expression of fluorescent and non-fluorescent proteins. Fluorescent proteins have uncovered transcriptional regulation, subcellular localization of proteins, and the fate of cells during development. Chromoproteins have received less attention until recent years as bioreporters. Here, we studied the properties of aeBlue, a a 25.91 kDa protein from the anemone Actinia equina. Objective: To assess the properties of aeBlue chromoprotein under different physicochemical conditions. Method: In this article, during the purification of aeBlue we uncovered that it suffered a color shift when frozen. We studied the color shift by different temperature incubation and physicochemical conditions and light spectroscopy. To assess the possible structural changes in the protein, circular dichroism analysis, size exclusion chromatography and native PAGE was performed. Results: We uncover that aeBlue chromoprotein, when expressed from a synthetic construct in Escherichia coli, showed a temperature dependent color shift. Protein purified at 4 °C by metal affinity chromatography exhibited a pinkish color and shifts back at higher temperatures to its intense blue color. Circular dichroism analysis revealed that the structure in the pink form of the protein has reduced secondary structure at 4 °C, but at 35 °C and higher, the structure shifts to a native conformation and Far UV- vis CD spectra revealed the shift in an aromatic residue of the chromophore. Also, the chromophore retains its properties in a wide range of conditions (pH, denaturants, reducing and oxidants agents). Quaternary structure is also maintained as a tetrameric conformation as shown by native gel and size exclusion chromatography. Conclusion: Our results suggest that the chromophore position in aeBlue is shifted from its native position rendering the pink color and the process to return it to its native blue conformation is temperature dependent.

>Magister Scientiae - MSc<br>Sea anemones (Order Actinaria) are a diverse order from the Class Anthozoa. They are found in all marine habitats at all depths and their symbiotic relationships play an important role in energy transfers especially in the benthic-pelagic community. The evolutionary background and phylogenetics of the class is poorly understood due to a lack of correspondence between taxonomic and molecular data (Daly et al. 2008). Therefore, a deeper exploration into Cnidarian molecular biology is needed to establish these as an evolutionary model organism. Gene discovery from various marine invertebrates has facilitated the recovery of anti-cancer drugs, antibiotics and reporter genes (Faulkner, 2000; Allen and Jaspars, 2009). The most commercially lucrative products from sea anemones are fluorescent and chromoproteins (FP/CP), which are used as non-invasive real-time reporter genes. The applications for these proteins are extensive and range from monitoring cellular processes such as protein localisation and interactions to imaging (Alieva et al. 2008). Therefore, novel FP and CPs have potential for commercialization. The aims of the project were to analyze basic molecular diversity of the sea anemones Pseudactinia varia, Pseudactina flagellifera and Bunodosoma capensis and evaluate a new screening method to isolate novel FP and CPs. To assess the basic molecular diversity, of the sea anemones and their associated symbionts 16S rRNA and 18S rRNA clone libraries were generated. The sea anemones used in this study clustered together with those of the Family Actiniidae. The bacterial associations observed based on the closest relative BLAST analysis were dominated by Proteobacteria (gamma, alpha and epsilon) as well as Bacteroides. The associate bacterial symbionts possibly produce compounds that range from polyunsaturated fatty acids, polyhydroxyalkanoates to anti-microbial compounds that aid the host in various processes. In order to screen for FPs and CPs from sea anemones three types of cDNA libraries were generated to be screened either by sequence based or activity based approaches. Novel primers were designed which could be applied for the screening of a variety of Anthozoans. A positive control was also designed and synthesised in order to test the capability of the designed primers and optimise the amplification. Although amplicons were generated from gDNA and cDNA libraries from each of the sea anemones they were found to be non-specific products. The detection limit is likely to be the limiting factor. The construction of an activity based library was not achieved due to technical constraints, which highlights the need for new molecular tools in this field or improvements to the existing ones.<br>National Research Foundation (NRF)

Unlike most other vertebrates, many species in the genus Etheostoma do not utilize structural refraction to display blue or green color. Instead, blue and green mating coloration exhibited by male rainbow darters (E. caeruleum) and male greenside darters (E. blennioides) results from the presence of true chromoprotein pigments. This study was conducted in order to extract, purify, characterize, and compare these novel pigments. Pigments were extracted in aqueous buffer and partially purified by ammonium sulfate fractionation and gel filtration chromatography. Final purification consisted of preparative non-denaturing polyacrylamide gel electrophoresis for E. caeruleum and hydroxyapatite chromatography for E. blennioides. Isolation of the chromophore was accomplished using acetone precipitation. The chromophore is the same in both species and is believed to be biliverdin. The protein component differs between the species and appears to have a greater number of subunits in E. blennioides. Binding of the protein to the chromophore amplifies the absorbance in the visible region and causes spectral tuning of the absorbance profile of the chromophore, with slight differences between species. In E. caeruleum, the chromoprotein pigment has a lambda max of 683 nm and transmits light at slightly shorter wavelengths, causing it to appear blue. In E. blennioides, the chromoprotein pigment has a lambda max of 696 nm and transmits light at slightly longer wavelengths, causing it to appear green. This work has shown that the protein component, not the chromophore, is responsible for the difference in hue between these two pigments. Future work will involve obtaining amino acid sequences for the protein component of the pigments and ultimately sequencing the gene coding for these proteins in darters.<br>Bayer School of Natural and Environmental Sciences;<br>Environmental Science and Management (ESM)<br>MS;<br>Thesis;

博士<br>國立臺灣大學<br>分子與細胞生物學研究所<br>103<br>GFP-like proteins have been studied with the aim of developing fluorescent proteins. Since the property of color variation is understudied, we isolated novel GFP-like chromoproteins from the carpet anemone Stichodactyla haddoni and Stichodactyla gigantea, termed shCP and sgBP, respectively. The shCP protein has maximum absorption wavelength peak (λmax) is located at 574 nm, resulting in a purple appearance. The sgBP has a λmax at 608 nm and shows a blue appearance. We mutated aa residues to enhance the color properties according to the simulated structure of shCP and the crystal structure of sgBP. With the mutations in and surrounding 5 Å of the chromophore, we found that the mutated proteins exhibited altered optical properties. The λmax of chromophore-mutated protein shCP-E63S was shifted to 560 nm and exhibited a pink color. shCP with surrounding mutations Q39S and T194I also exhibited coloration changes to red and purple-blue. An additional mutation at I196H of the mutated protein shCP-E63L exhibited green fluorescence. In the chromoprotein sgBP, the surrounding S157C mutation shifted the λmax to 604 nm and darkened the blue color expression, indicating the color expression of chromoprotein could be altered by deprotonation state change of the phenolic group in the chromophore. Additionally, we found that the cDNAs of shCP, sgBP and its mutated varieties are faithfully and stably expressed both in Escherichia coli and zebrafish embryos. Our results provide a structural basis for the color regulation of the biomarker development.

博士<br>國立中興大學<br>化學系所<br>100<br>Neocarzinostatin is a natural antitumor antibiotic chromoprotein complex. Its carrier protein protects labile enediyne chromophore and regulates its release. When the chromophore is unbound, its enediyne moiety can be easily cycloaromatized by thiols. When the chromophore is protein-bound, the carrier protein diverts cycloaromatization to a different pathway. Our previous findings suggested that the disulfide bonds might play a key role in directing the cycloaromatization pathway. We replaced cysteine residues with serine, and found that rupture of the disulfide bond Cys37-Cys47, which maintains steric rigidity of the binding cavity, caused the loss of chemical directing ability, but rupture of the disulfide bond Cys88-Cys93 had only a minor influence. How the microorganism preserves the secreted antibiotic and at the same time is immune to its toxicity are interesting issues. Neocarzinostatin is produced by Streptomyces carzinostaticus. Biothiol analyses revealed that cells of Streptomyces carzinostaticus produced no acidic glutathione, but instead neutral mycothiol. When bacteria were cultured under a condition that stimulated the biosynthesis of neocarzinostatin, the yield of mycothiol increased significantly. Incubating neocarzinostatin with cell extract that retained active mycothiol led to efficient drug inactivation. The anionic side chains of D33 and D99 in the neocarzinostatin protein played two critical roles in a single thiol-screening operation: preserving the antibiotic for defense and survival by rejecting the ubiquitous glutathione through charge-charge repulsion in the outer-cell environment, and detoxifying the toxin in the inner-cell body for self-resistance by accepting the cell-produced neutral mycothiol. We employed mutations on residues around the binding cleft to study chemistry of the protein-directed cycloaromatization pathway, and found that residue D79 played an important role. We proposed a mechanism and discussed the influence of the negatively charged side chain on the protein-directed cycloaromatization pathway. The side chain of F78, as demonstrated by this lab, plays a role in gating the chromophore release. Here we repeated the kinetic study of chromophore-releasing by using mutants at F78. The results showed that mutants F78R and F78K efficiently stimulated the chromophore release, whereas F78Y and F78W had a release rate close to that of the wild-type. In conclusion, neocarzinostatin protein plays important roles in the cycloaromatization, protection and release of its chromophore. We hope that these results could provide useful information for clinical design of the drug.

博士<br>國立中興大學<br>化學系<br>94<br>Neocarzinostatin, the antitumor antibiotic chromoprotein from Streptomyces carzinostaicus, is a non-covalent complex of a highly stable all β-sheet apoprotein of 113 amino acids and a labile 9-membered enediyne chromophore. The apoprotein stabilize and transports its chromophore, also regulates its release in juxtaposition to the target DNA. The present study is intended to understand (i) the mechanism of the release of the chromophore from the chromoprotein complex; (ii) the structural stability of the carrier protein; and (iii) the mechanism of sodium chloride inhibition of DNA cleavage induced by neocarzinostatin. The first chapter of the thesis, the “Introduction”, is an elaborate account describing the system and the objectives along with reviewing the literatures dealing with the discovery, classification, structure, and mechanism of activity of enediyne antitumor antibiotics, with particular references to neocarzinostatin. The second chapter deals with the expression, purification, and preliminary biophysical characterizations of the recombinant aponeocarzinostatin. The third chapter describes the mechanism of the 2,2,2-trifluoroetanol-induced release of the chromophore. The studies involve circular dichroism, fluorescence, HPLC, and 1-dimensional nuclear magnetic resonance analyses of aponeocarzinostatin and the chromophore release. The results show that the release of chromophore occurs before the unfolding of aponeocarzinostatin becomes evident, suggesting that the release does not require a major conformational change in the protein. The fourth chapter describes the exploration of the natural driving force(s) underlying the chromophore release. The studies involved structural changes of aponeocarzinostatin and release kinetics of the chromophore in the presence of organic solvents (alcohols and acetonitrile) and salts. The results, in concert, suggest that the release of the chromophore, which precedes major conformational changes of aponeocarzinostatin induced by organic solvents, is caused by hydrophobic perturbation, rather than ionic. The fifth chapter describes experiments to assess the stability of aponeocarzinostatin against chemical denaturants and organic solvents, since the structural stability is an important requirement for being an efficient proteinaceous drug carrier. While aponeocarzinostatin retains almost its native structure in fairly high concentrations of organic solvents, it shows only partial unfolding at the maximum concentration of urea and guanidine hydrochloride. It also retains its structural integrity in the potent denaturant sodium n-dodecyl sulfate. The structural stability of aponeocarzinostatin against organic solvents and chemical denaturants is higher than that of cytochrome c, lysozyme, and several other proteins which are known to be stable. The comparison among proteins provides evidence to the unusually high stability of aponeocarzinostatin. The results highlight the appropriateness of aponeocarzinostatin to protect its labile chromophoric partner. The sixth chapter presents an attempt to understand the mechanism of salt inhibition of neocarzinostatin-mediated DNA strand scission, an unexplored aspect of neocarzinostatin activity. Earlier studies on neocarzinostatin elsewhere showed that sodium chloride inhibits the neocarzinostatin-mediated DNA strand scission. Our data from in vitro DNA cleavage experiments under different sodium chloride concentrations, performed in the presence of thiols (β-mercaptoethanol or glutathione), show that the salt-protein interaction is not be the major inhibitory factor. There is little effect of sodium chloride on the thermal stability of aponeocarzinostatin. Fluorescence analysis of release kinetics of the chromophore reveals little influence of salt on the rate of release. The DNA binding data suggest that the major inhibitory effect of salt on DNA cleavage is caused by preventing the binding of the chromophore with the target DNA.

Tese de Doutoramento em Engenharia Química e Biológica<br>The inflammatory response is an important step in wound healing, however if there is an imbalance between the immune response and tissue regeneration, a delay on the healing process will occur, resulting in a chronic wound. Chronic wounds are a significant problem for health care system, accounting for almost 4% of its total budget, and currently increasing. Early detection of incipient wound infection and chronic inflammation reduces the severity of the disease and decreases health care expenses. Currently, it is difficult, expensive and time consuming to accurately assess a wound status, hence, there is an urgent need for a new diagnostic that would give the medical staff a fast and reliable method to determine inflammation/infection of a chronic wound at an early stage. Human neutrophil elastase (HNE) is one of the most abundant proteinases in wounds and its activity was found to be elevated in case of infection and chronic inflammation, making this enzyme a suitable marker for impaired healing. This PhD thesis intends to explore new strategies for the detection of HNE through the development of chromogenic and fluorogenic sensors that when embedded in a dressing may provide an in situ and real-time assessment of the wound status. For that, a specific cleavage sequence for HNE was designed by molecular docking studies and then confirmed in vitro. Then, this substrate was associated to chromogenic or fluorogenic detection systems. Regarding the chromogenic detection systems, two strategies were followed using ultramarine (UM), a GFP-like chromoprotein. The rationale of this approach was to use this protein’s ability to regenerate its dark blue colour (hysteresis) upon HNE cleavage, turning it into a switch-on sensor, or to lose its colour upon proteolysis, thus making a switch-off sensor. In a first attempt to convert ultramarine into a HNE substrate switch was based on a split protein strategy which would signal as a gain of colour sign. We were able to detect an absorbance increase upon HNE cleavage, but this effect was not long lasting due to an instability of the interaction between the signalling and sensing fragments. Indeed, this strategy proved it to be extremely challenging. The main motivation to pursue this work resided in its innovative aspect, as GFP-like chromogenic sensors were never reported. Then, a second strategy was investigated through sitedirected mutagenesis of ultramarine. In this latter strategy we were able to obtain sensors with an opposite signal, switch-off. To provide a better understanding regarding UM’s chromogenic environment, molecular dynamics studies coupled to protein expression were implemented to each of the seven produced UM-mutants. Our findings provided new information regarding the effect of certain mutations in UM protein conformation and allowed us to explore its application as a switch sensor. Here, we explored for the first time the development of a colour switch-on sensor based on chromogenic GFP-like proteins. Even though, we were unable to fully accomplish the regeneration of a stable chromophore environment for colour appearance, this work may serve as a starting point for advances in switch-on chromogenic sensors using non-fluorescent GFP-like proteins. For the fluorogenic detection system, the strategy studied resulted in the successful development of a FRET (Förster resonance energy transfer) peptide to monitor HNE. Upon HNE action, this peptide proved to be more effective than the traditional peptides coupled to fluorophores. Its visual detection was possible using an UV-portable light source. The embedding of this sensor in dressings would provide advantages for its application as a wearable sensor. The methods here used for the FRET-peptide immobilization onto a dressing compromised its detection. However, we envision that with the proper method of integration into a dressing, this FRET peptide could be used as a new strategy of smart materials. The strategies explored in this thesis can be further investigated and possibly implemented as point-of-care medical devices for wound control. As wound care tools, these devices would allow a prompt detection of chronic wounds, contributing to their proper and effective care and, consequently, granting a better outcome for the millions of people that suffer from non-healing wounds.<br>A resposta inflamatória é um importante passo no processo de cicatrização de feridas. No entanto, desequilíbrios entre a resposta do sistema imunitário e a regeneração celular podem desencadear um atraso significativo no processo de cicatrização, resultando numa ferida crónica. As feridas crónicas apresentam um problema significativo para o sistema de saúde, contabilizando 4% do orçamento total de saúde, sendo que se estima um aumento destes números nas próximas décadas. A deteção antecipada de infeção e inflamação crónica numa ferida reduz a severidade da doença, consequentemente diminuindo o seu impacto nas despesas de saúde. Atualmente, a avaliação do estado de uma ferida de forma exata e precisa é um processo difícil, dispendioso e demoroso. Por esta razão, existe uma necessidade urgente de desenvolvimento de novas ferramentas de diagnóstico que possibilitem a deteção precoce de infeção/inflamação em feridas crónicas de forma rápida e eficaz. A elastase neutrofílica humana (HNE) é uma das proteinases mais abundantes em feridas, estando os seus elevados níveis de atividade em feridas correlacionados com a ocorrência de infeção e inflamação crónica. De facto, esta enzima é considerada um excelente biomarcador para feridas cujo processo de cicatrização se encontra comprometido. O objetivo principal desta tese de doutoramento foi e exploração de novas estratégias para a deteção da HNE através do desenvolvimento de sensores cromogénicos e fluorogénicos que possam ser incorporados em pensos para feridas, possibilitando uma avaliação in situ e em temporeal do estado da ferida. Para tal, primeiramente desenharam-se sequências de clivagem específicas para HNE cuja afinidade para a enzima foi analisada através de estudos de acoplamento molecular e posteriormente foram confirmados com ensaios in vitro. Depois, o substrato foi associado a sistemas de deteção cromogénico ou fluorogénico. Relativamente aos sistemas de deteção cromogénicos, desenvolveram-se duas estratégias usando a ultramarina (UM), uma cromoproteína do tipo GFP, como substrato sinalizador. A competência desta proteína em regenerar a sua cor azul (devido à capacidade de histerese) permitiu transformá-la num sensor que se liga (switch-on) ou que se desliga (switch-off) após a sua clivagem com a HNE. A primeira tentativa para converter a proteína ultramarina num substrato comutador de sinal para a HNE consistiu na produção de uma proteína segmentada em duas partes que quando em interação resultam num sinal com ganho de cor (sensor switch-on). Após clivagem com HNE do fragmento substrato, conseguiu-se obter um aumento da absorvância, embora transiente, devido à interação com o fragmento sinalizador. De facto, esta estratégia foi muito desafiante. No entanto, a principal motivação em seguir esta linha de trabalho prendeu-se principalmente com o seu carácter inovador, visto que nunca tinham sido reportados sensores usando proteínas cromogénicas do tipo GFP. Neste seguimento, explorou-se uma segunda estratégia para o desenvolvimento de substratos sensores switch-on para a HNE através da mutagénese direcionada da proteína ultramarina. Esta abordagem permitiu também obter sensores com o sinal oposto – switch-off. Com o objetivo de melhor compreender as condições que proporcionam o ambiente cromogénico da proteína UM, foram implementados estudos de simulação de dinâmica molecular complementares à expressão das proteínas para cada um dos sete mutantes produzidos. A partir deste trabalho conseguiu-se recolher novos dados acerca do efeito de certas mutações na conformação da proteína UM e possibilitou-nos também explorar a sua aplicação como sensor comutador de sinal. Nestes trabalhos investigou-se pela primeira vez o desenvolvimento de sensores cromogénicos switch-on baseados em proteínas do tipo GFP. Embora não se tenha conseguido obter a regeneração estável do cromóforo que permite o aparecimento de cor, estes resultados poderão servir como ponto de partida para outros trabalhos em sensores cromogénicos switch-on usando proteínas não fluorescentes do tipo GFP. Em relação aos sistemas de deteção fluorogénicos, a abordagem explorada resultou no desenvolvimento bem-sucedido de um péptido FRET para monitorização de HNE. Após proteólise, este péptido provou ser mais eficaz do que os tradicionais péptidos fluorescentes (acoplados a fluoróforos). A deteção visual da fluorescência foi possível recorrendo a uma luz UV portátil. A incorporação deste sensor em pensos de feridas trará vantagens relativamente à sua aplicação como sensores wearable. Os métodos aqui descritos para a imobilização do péptido FRET em têxteis usados como material de penso comprometeram a deteção do seu sinal. No entanto, prevêse que a utilização de um método de integração mais apropriado possa permitir a sua aplicação em novas estratégias no desenvolvimento de tecidos inteligentes. A implementação da investigação aqui descrita em dispositivos médicos para a monitorização do processo de cicatrização de feridas poderá complementar o leque de ferramentas usadas no cuidado das feridas. Estes dispositivos possibilitarão a deteção atempada da cronicidade de uma ferida, contribuindo para o seu cuidado apropriado e eficaz. Consequentemente irá garantir uma melhor qualidade de vida para milhões de doentes que sofrem de feridas de difícil cicatrização.<br>Fundação para a Ciência e Tecnologia (FCT) e à Tecminho pelo financiamento da minha bolsa de doutoramento (SFRH/BD/113247/2015). Ao projeto da comissão europeia INFACT (FP7-NMP-2013-SME-7 - Grant agreement no. 604278)