566 research outputs found
Using a reprogrammed genetic code to modulate protein activity by novel post-translational control
Despite the diverse structures and functions sampled by the proteome, all proteins comprise 20 canonical amino acids that sample only a small percentage of available chemistry. This limitation is lifted somewhat through the use of post-translational modifications, however the limit imposed by the restricted number of amino acids inherently limits the variety of protein function and control that can be accessed. One powerful route to diversify the chemistry sampled by proteins is through genetically encoded unnatural amino acid (uAA) incorporation. The uAA p-azido-L-phenylalanine (AzPhe) can introduce two novel methods of control, photochemical covalent rearrangement and Click chemistry. AzPhe incorporation combined with these two methods of novel post-translational control were used to modulate the function of two distinct proteins; TEM β-lactamase and sfGFP.
This thesis introduces the use of uAAs and the technical modifications required to enable uAA incorporation in vivo. It describes the in silico approach taken to evaluate potential mutations based on the likelihood of them imparting novel changes to protein function. Nine positions in TEM β-lactamase were chosen for uAA incorporation and the effect on activity was then determined using kinetic analyses. AzPhe incorporation alone resulted in a variety of effects on enzyme activity, ranging from small increases to complete loss of activity. Subsequent post-translational modification using UV light resulted in only slight changes in activity. Modification via Click chemistry using dibenzyl cyclo-octyne (DBCO) derivatives resulted in either inhibition or increased catalytic activity, depending on the position of AzPhe incorporation and the type of adduct used.
Click chemistry was then used to modify TEM β-lactamase with other chemical modifications that enable the immobilization of proteins onto two different surfaces. The π-π stacking interaction between a DBCO-pyrene moiety and graphene was exploited to attach TEM β-lactamase to graphene in a defined and controlled manner, placing the active site in close proximity to the electron cloud of the sp2-bonded material. TEM β-lactamase was then modified using two DNA oligonucleotides that define assembly of a DNA origami “tile”. DNA origami can be used to immobilize multiple proteins at several defined positions, enabling the re-creation of enzyme pathways or signalling cascades in vitro.
Finally, AzPhe was incorporated into sfGFP and the effects of its incorporation and subsequent modification on fluorescence were explored. The incorporation of AzPhe resulted in a blue shifted λmax, a change that was reversed upon UV irradiation. X-ray crystallography suggested that a hydrogen-bonding network involving the chromophore and surrounding residues was disrupted upon AzPhe incorporation, but then reformed upon modification of the uAA. Click chemistry had a variable effect on fluorescence depending on the modification used. Modification of AzPhe with a large fluorescent dye had no effect on the sfGFP fluorescence spectrum, but enabled FRET between the two chromophores. Modification with a DBCO-amine had the same effect as UV irradiation.
Overall, this thesis has shown that the use of genetically encoded uAA incorporation coupled with novel post-translational modifications is a powerful approach for modifying protein function, and facilitating defined interfacing with new and useful materials
The Development of Attitudes Toward Scientific Models During a Participatory Modeling Process – The Impact of Participation and Social Network Structure
Scientific models are increasingly being used to support participatory natural resources management decision making processes. These models allow stakeholders and scientists to explore potential policy and management options and can help facilitate discussion surrounding concerning uncertainty and different sources of knowledge. The unique benefits of participatory modeling processes, however, are contingent upon stakeholders understanding of, engagement with, and willingness to use the scientific models as sources of knowledge and information. Little is known, however, about how stakeholders view scientific models within these processes. We examined changes in stakeholders’ attitudes toward scientific models over the course of OysterFutures, a 2-year, facilitated participatory modeling process that aimed to create consensus recommendations for oyster management in the Choptank River Complex, MD, United States. Five ordered logistic regression models were used to test hypotheses concerning the impact of social network measures, factors related to the participatory modeling process itself, and stakeholder characteristics on salience, credibility and legitimacy (SCL) attitudes toward models. Results suggested that stakeholders’ ways of knowing was a significant driver of salience, credibility and legitimacy elements of attitudes toward models. Additionally, acting as a gatekeeper within the social network resulted in significantly lower attitudes toward model credibility. These results indicate that the scientific model acted as a boundary object that facilitated discussion during the participatory modeling process. By better understanding the factors that influence model attitude formation, these processes can adjust their design and function to better take advantage of these models. Additionally, practitioners can have more realistic expectations concerning the role of models within participatory, collaborative natural resources decision-making processes
Single Synonymous Mutations in KRAS Cause Transformed Phenotypes in NIH3T3 Cells
Synonymous mutations in the KRAS gene are clustered at G12, G13, and G60 in human cancers. We constructed 9 stable NIH3T3 cell lines expressing KRAS, each with one of these synonymous mutations. Compared to the negative control cell line expressing the wild type human KRAS gene, all the synonymous mutant lines expressed more KRAS protein, grew more rapidly and to higher densities, and were more invasive in multiple assays. Three of the cell lines showed dramatic loss of contact inhibition, were more refractile under phase contrast, and their refractility was greatly reduced by treatment with trametinib. Codon usage at these glycines is highly conserved in KRAS compared to HRAS, indicating selective pressure. These transformed phenotypes suggest that synonymous mutations found in driver genes such as KRAS may play a role in human cancers
Interpreting the seasonal cycles of atmospheric oxygen and carbon dioxide concentrations at American Samoa Observatory
We present seven years of atmospheric O2/N2 ratio and CO2 concentration data measured from flask samples collected at American Samoa. These data are unusual, exhibiting higher short-term variability, and seasonal cycles not in phase with other sampling stations. The unique nature of atmospheric data from Samoa has been noted previously from measurements of CO2, methyl chloroform, and ozone. With our O2 data, we observe greater magnitude in the short-term variability, but, in contrast, no clear seasonal pattern to this variability. This we attribute to significant regional sources and sinks existing for O2 in both hemispheres, and a dependence on both the latitudinal and altitudinal origins of air masses. We also hypothesize that some samples exhibit a component of "older" air, demonstrating recirculation of air within the tropics. Our findings could be used to help constrain atmospheric transport models which are not well characterized in tropical regions
Separation of nickel from cobalt and manganese in lithium ion batteries using deep eutectic solvents
The authors would like to thank the Faraday Institution (grant codes FIRG005 and FIRG006) for funding (Project website https://relib.org.uk). This research also received funding from the European Commission's H2020 – Marie Sklodowska Curie Actions (MSCA) − Innovative Training Networks within the SOCRATES project under the grant agreement no. 721385 (Project website: https://etn-socrates.eu).A cornerstone of the decarbonisation agenda is the use of lithium ion batteries, particularly for electric vehicles. It is essential that effective recycling protocols are developed and this includes the ability to selectively digest and recover components of the cathode materials, most commonly including manganese, cobalt and nickel. This study shows a method by which nickel oxide can be efficiently separated from cobalt and manganese oxides using an oxalic acid-based deep eutectic solvent. The subsequent addition of water to the pregnant solution enables the co-precipitation of cobalt and manganese oxalates. This permits a route to the reformulation of the active materials from high cobalt and manganese content to high nickel content.Publisher PDFPeer reviewe
Molecular basis for functional switching of GFP by two disparate non-native post-translational modifications of a phenyl azide reaction handle
Through the genetic incorporation of a single phenyl azide group into superfolder GFP (sfGFP) at residue 148 we provide a molecular description of how this highly versatile chemical handle can be used to positively switch protein function in vitro and in vivo via either photochemistry or bioconjugation. Replacement of H148 with p-azido-L-phenylalanine (azF) blue shifts the major excitation peak ∼90 nm by disrupting the H-bond and proton transfer network that defines the chromophore charged state. Bioorthogonal click modification with a simple dibenzylcyclooctyne or UV irradiation shifts the neutral-anionic chromophore equilibrium, switching fluorescence to the optimal ∼490 nm excitation. Click modification also improved quantum yield over both the unmodified and original protein. Crystal structures of both the click modified and photochemically converted forms show that functional switching is due to local conformational changes that optimise the interaction networks surrounding the chromophore. Crystal structure and mass spectrometry studies of the irradiated protein suggest that the phenyl azide converts to a dehydroazepine and/or an azepinone. Thus, protein embedded phenyl azides can be used beyond simple photocrosslinkers and passive conjugation handles, and mimic many natural post-translational modifications: modulation though changes in interaction networks
MitoNeoD:a mitochondria-targeted superoxide probe
Mitochondrial superoxide (O2⋅−) underlies much oxidative damage and redox signaling. Fluorescent probes can detect O2⋅−, but are of limited applicability in vivo, while in cells their usefulness is constrained by side reactions and DNA intercalation. To overcome these limitations, we developed a dual-purpose mitochondrial O2⋅− probe, MitoNeoD, which can assess O2⋅− changes in vivo by mass spectrometry and in vitro by fluorescence. MitoNeoD comprises a O2⋅−-sensitive reduced phenanthridinium moiety modified to prevent DNA intercalation, as well as a carbon-deuterium bond to enhance its selectivity for O2⋅− over non-specific oxidation, and a triphenylphosphonium lipophilic cation moiety leading to the rapid accumulation within mitochondria. We demonstrated that MitoNeoD was a versatile and robust probe to assess changes in mitochondrial O2⋅− from isolated mitochondria to animal models, thus offering a way to examine the many roles of mitochondrial O2⋅−production in health and disease
Selective superoxide generation within mitochondria by the targeted redox cycler MitoParaquat
Superoxide is the proximal reactive oxygen species (ROS) produced by the mitochondrial respiratory chain and plays a major role in pathological oxidative stress and redox signaling. While there are tools to detect or decrease mitochondrial superoxide, none can rapidly and specifically increase superoxide production within the mitochondrial matrix. This lack impedes progress, making it challenging to assess accurately the roles of mitochondrial superoxide in cells and in vivo. To address this unmet need, we synthesized and characterized a mitochondria-targeted redox cycler, MitoParaquat (MitoPQ) that comprises a triphenylphosphonium lipophilic cation conjugated to the redox cycler paraquat. MitoPQ accumulates selectively in the mitochondrial matrix driven by the membrane potential. Within the matrix, MitoPQ produces superoxide by redox cycling at the flavin site of complex I, selectively increasing superoxide production within mitochondria. MitoPQ increased mitochondrial superoxide in isolated mitochondria and cells in culture ~a thousand-fold more effectively than untargeted paraquat. MitoPQ was also more toxic than paraquat in the isolated perfused heart and in Drosophila in vivo. MitoPQ enables the selective generation of superoxide within mitochondria and is a useful tool to investigate the many roles of mitochondrial superoxide in pathology and redox signaling in cells and in vivo
Association between Depressive Symptoms and Metabolic Syndrome in Police Officers: Results from Two Cross-Sectional Studies
Policing is one of the most dangerous and stressful occupations and such stress can have deleterious effects on health. The purpose of this study was to examine the association between depressive symptoms and metabolic syndrome (MetSyn) in male and female police officers from two study populations, Buffalo, NY and Spokane, WA. Depressive symptoms were measured using the Center for Epidemiologic Studies-Depression (CES-D) scale. MetSyn was defined using the 2005 AHA/NHBLI guidelines. Analysis of covariance was used to describe differences in number of MetSyn components across depressive symptom categories. The number of MetSyn components increased significantly across categories of CES-D for Spokane men only (p-trend = 0.003). For each 5-unit increase in CES-D score, odds increased by 47.6% for having hypertriglyceridemia, by 51.8% for having hypertension, and by 56.7% for having glucose intolerance. Exploring this association is important since both are predictors of future chronic health problems and the results could be helpful in developing future gender-specific prevention and intervention efforts among police officers
A comparison of photocatalytic reforming reactions of methanol and triethanolamine with Pd supported on titania and graphitic carbon nitride
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