No description available.

No description available.

The interactions between protein-protein, protein-nucleic acid, and protein-small molecules are central to biological processes and are key for the design of new therapeutics. Rapid and easy to implement methodologies are needed that enable the interrogation of these interactions in a complex cellular context. Towards this goal, I have utilized the concept of split-protein reassembly, also called protein complementation, for the creation of a variety of sensor architectures that enable the interrogation of protein-nucleic acid, protein-protein, and protein-small molecule interactions. Utilizing the enzymatic split-reporter β-lactamase and existing zinc finger design strategies we applied our recently developed technology termed SEquence-Enabled Reassembly (SEER) towards the creation of a sensor capable of the specific detection of the CryIA transgene. Additionally, the split β-lactamase reporter was utilized for the sitespecific determination of DNA methylation at cytosine residues that is involved in epigenetic regulation. This method, dubbed mCpG-SEER, enabled the direct detection of femtomole levels of dsDNA methylation in sequence specific manner. In a separate endeavor, we have developed and optimized the first cell-free split-reporter systems for GFP, split β-lactamase, and firefly luciferase for the successful dsDNA-dependent reassembly of the various reporters. Our cell free in vitro translation systems eliminates previous bottlenecks encountered in split-reporter technologies such as laborious transfection/cell culture or protein purification. Capitalizing on the ease of use and speed afforded by this new technology we describe the sensitive detection of protein-protein, protein-nucleic acid, and protein-small molecule interactions and inhibitors thereof. In a related area, we have applied this rapid cell-free split-firefly luciferase assay to the specific interrogation of a large class of helix-receptor protein-protein interactions. We have built a panel consisting of the clinically relevant Bcl-2 family of proteins, hDM2, hDM4, and p53 and interrogated the specificity of helix-receptor interactions as well as the specificity of peptide and small-molecule inhibitors of these interactions. Finally, we describe the further applications of our cell-free technology to the development of a large number of general split-firefly luciferase sensors for the detection of ssRNA sequences, the detection of native proteins, the evaluation of protease activity, and interrogation of enzyme-inhibitor interactions. The new methodologies provided in this study provides a general and enabling methodology for the rapid interrogation of a wide variety of biomolecular interactions and their antagonists without the limitations imposed by current in vitro and in vivo approaches.

ACCURACY OF TRANSFER RNA SELECTION IN PROTEIN SY The ribosome is a rapid magnificent molecular machine that plays an important role in proteinsynthesis and it consists of RNA and protein. The 70S bacterial ribosome comprises twosubunits, 30S and 50S. The 30S small subunit of the bacterial ribosome contains a protein calledS12, encoded by the rpsL gene. The function of this S12 protein is to help arrange the mRNAcorrectly to the ribosome and to interact with transfer RNA (tRNA) to initiate translation.Mutations in the rpsL gene generate phenotypes like resistance, dependence or pseudodependenceto the antibiotic streptomycin in bacteria. It is believed that mutations in the rpsLgene can increase the accuracy of tRNA selection in protein synthesis.The ribosome conducts the selection of tRNA in two steps: the initial selection and theproofreading step. During these multiple steps, the ribosome chooses the cognate aminoacyltRNAsin a ternary complex with EF-Tu and GTP and accommodates in the A site of ribosome.Therefore, the accuracy of the ribosome in selection of cognate aminoacyl-tRNA is crucial for the production of functional polypeptide sequences. Here, three different Escherichia coli strains; wild type MG1655, streptomycin restrictive (SmR) strain res222, and a streptomycin pseudo-dependent (SmP) strain w3110 are used, for studying the accuracy of tRNA selection inprotein synthesis. The mutant SmR shows hyper-accurate phenotype, which means, it has lowerpeptide bond formation efficiency and higher accuracy than the wild type. SmP shows pseudodependentto streptomycin phenotype which means it has higher peptide bond formation efficiency in the presence of antibiotic streptomycin. I have estimated the accuracy of tRNA selection in protein synthesis with enzyme kinetics. The kinetics data of these experiments display that mutant streptomycin restrictive is hyper-accurate and lower peptide bond formation efficiency than the wild type. SmP for the near cognate reaction in presence of antibiotic streptomycin has higher peptide bond formation efficiency than the SmP in absence of antibiotic streptomycin. SmP in presence antibiotic streptomycin has lower accuracy than the SmP in absence of antibiotic streptomycin.

Protein-lipid complexes are known to result from complex molecular interactions which contribute to physiochemical and functional properties of foods. To identify the interactions of food proteins with lipids and the associated changes in properties, the following factors were investigated: incubation temperature, pH, type of lipids (phospholipid, triglyceride and fatty acids) and different proteins (ovalbumin and soybean glycinin). The effects of lipids on physiochemical and functional characteristics of ovalbumin and glycinin were investigated using polyacrylamide gel electrophoresis (PAGE), fluorescence, differential scanning calorimetry (DSC), Fourier transform infrared (FTIR) spectroscopy, and emulsification and gelation properties. / The results showed that pH, temperature and the type of lipids affected both ovalbumin-lipid interactions and physiochemical properties of ovalbumin. Changes in the electrophoretic behavior of ovalbumin were related to the presence of lipids, and the relative fluorescence of ovalbumin decreased in the presence of lipids at different pH values. In addition, lipids increased the stability of ovalbumin as revealed by the thermal denaturation (Td) and by the enthalpy transition (DeltaH). FTIR spectra in the amide I absorption region revealed that lipids affected the secondary structure of ovalbumin. Changes in the integrated intensity of the amide II band between (1520--1555) cm -1 in the presence of D2O showed that H-D exchange of ovalbumin decreased in the presence of lipids. Emulsifying properties, gel strength and water holding capacity (WHC) of ovalbumin increased significantly (P < 0.05) in the presence of lipids. Scanning electron microscopy (SEM) showed difference in the microstructure of ovalbumin gel in the presence of different lipids. The more pronounced effect of lipids was observed with lecithin and the lowest was with stearic acid. The order of magnitude for the effects of lipids on physiochemical and functional properties of ovalbumin was: lecithin > cocoa butter > oleic acid > linoleic acid > linolenic acid > stearic acid. It is likely that the degree of polarity of lipids play an important role in protein-lipid interactions and in the enhancement of the functional properties of ovalbumin. / The effects of soybean oil (SO), soybean lecithin (SL) and a mixture of both (SOL) on physiochemical and functional properties of soybean glycinin were studied at pH 8.0, with incubation at 40°C. Changes in the electrophoretic behavior were related to the presence of lipids. Relative fluorescence of glycinin decreased in the presence of lipids. Lipids increased the thermal stability (Td) of glycinin from 89.7°C to 92.0°C, 94.3°C and 93.4°C with SO, SL and SOL respectively. FTIR spectra indicated changes in both; the secondary structure and H-D exchange of glycinin in the presence of lipids. Gel strength, WHC and emulsifying properties of glycinin increased significantly (P < 0.05) in the presence of lipids; the order of consequence increase was: SL > SOL > SO. SEM showed difference in the microstructure of glycinin gels with the different lipids used. Overall, the results demonstrate both quantitative and qualitative effects on the physiochemical and functional properties of ovalbumin and glycinin as a result of protein-lipid interactions.

No description available.

No description available.

Copper-containing nitrite reductase (NiR) is a homotrimer of two cupredoxin domains and catalyzes the single electron reduction of NO2- to NO during dissimilatory denitrification. To investigate the evolution of NiR, methods of mutagenic library generation and high-throughput variant screening from E. coli colonies were developed. These methods allow for facile screening of 105 mutants for folding efficiency or substrate specificity. Initial proof of principle studies yielded several variants that oxidized the artificial substrate ο-dianisidine up to 8 times faster than wild type NiR, suggesting that this methodology has the potential to engineer NiR to acquire other reductase functions. A crystal structure was solved for a putative multicopper oxidase (MCO) and NiR homologue from Arthrobacter sp. (AMMCO) to 1.8 Å resolution. The overall folds of AMMCO and NiR are very similar (r.m.s.d. of 2.0 Å over 250 Cα atoms); Like NiR, AMMCO is a trimer with type-1 Cu sites in the N-terminal domain of each monomer; however, the active site of AMMCO contains trinuclear Cu site characteristic of MCOs instead of a the mononuclear type-2 Cu site found in NiR. Detailed structural analysis supports the theory that two-domain MCOs similar to AMMCO were intermediaries in the evolution of NiR and the more common three-domain MCOs. The physiological function of AMMCO remains uncertain, but genomic, crystallographic and functional analysis suggests that the enzyme is involved in metal regulation. Considering the extensive similarity between AMMCO and NiR, particularly at the active site, engineering a trinuclear cluster into NiR appears feasible with a modest number of alterations to the polypeptide chain. With the aid of my newly developed high-throughput screening technique and site-directed mutagenesis, the mononuclear NiR active site was remodelled into a trinuclear Cu site similar to that of MCO. A crystal structure of this variant was solved to 2.0 Å and the presence of three copper atoms at the engineered cluster was confirmed by Cu-edge anomalous diffraction data. Although the trinuclear copper cluster is present and catalyzes the reduction of oxygen, achieving rates of catalysis seen in native MCOs has proven more difficult. With the framework provided, further engineering NiR into a robust MCO is likely to provide further insights into the structural basis of oxygen reduction by trinuclear copper sites.

Protein synthesis (mRNA) is tightly regulated under numerous conditions in cardiomyocytes. It can be activated by hormones such as insulin and also by other agents such as phenylephrine (PE) that activates hypertrophy in the heart. Cardiac hypertrophy involves an increase in the muscle mass of the heart, principally in the left ventricular muscle, and the increase is due to enlarged cell size, not increased cell number. A pivotal element of cardiac hypertrophy is an elevation in the rates of protein synthesis, which drives the increase in cell size causing hypertrophy. Unfortunately, we currently lack the understanding of the basic mechanisms that drives hyperactivated protein synthesis. Cardiac hypertrophy is clinically important because it is a major risk factor for heart failure. It initially serves as an adaptive response to increase cardiac output in response to higher demand, but ultimately leads to deterioration of contractility of the heart if hypertrophy is sustained. The main goal of this research project is to understand how hypertrophic agents, such as phenylephrine (PE), activate protein synthesis using adult rat ventricular cardiomyocytes as a model. Specifically, this study focuses on how the translational initiation is controlled by upstream signalling pathways.

19F NMR studies of proteins provide unique insight into biologically relevant phenomena such as conformational fluctuations, folding and unfolding, binding and catalysis. While there are many advantages to the use of 19F NMR, experimental challenges limit its widespread application. The focus of this thesis has been to address some of these limitations, including resonance assignment and perturbations arising from fluorine probes, and to develop more robust methods of studying protein topology by 19F NMR. 19F NMR experiments designed to measure local hydrophobicity and exposure were developed and evaluated in two systems, Fyn SH3 and calmodulin, labeled with 3-fluorotyrosine. Paramagnetic effects from dissolved oxygen, solvent isotope shifts from deuterium oxide, and 1H-19F NOEs were each sufficient in establishing relative solvent exposure, while the combination of effects from oxygen and deuterium oxide were able to delineate local hydrophobicity and solvent accessibility of 19F probes. Two NMR based resonance assignment protocols were developed using 13C, 15N-enriched 3-fluorotyrosine and 3-fluorophenylalanine, separately biosynthetically incorporated into calmodulin. In the first approach, isotopic enrichment facilitated two-dimensional heteronuclear experiments based on INEPT and COSY magnetization transfer schemes to correlate the fluorine nucleus to sidechain and backbone 1H, 13C, and 15N atoms, providing complete spectral assignment. The assignment of 3-fluorophenylalanine resonances was achieved using 19F-, and 15N-edited homonuclear NOE experiments to connect the fluorine nucleus to intraresidue and neighboring 1H and 15N resonances. While both strategies were successful, the NOE-based method was vulnerable to alternate relaxation mechanisms, including chemical shift anisotropy and chemical exchange. Structural perturbations arising from uniform incorporation of 3-fluorophenylalanine in calmodulin was thoroughly investigated using 19F and 1H-15N NMR spectroscopy, 15N spin relaxation and thermal denaturation via circular dichroism spectroscopy. While stability was unaffected, NMR experiments revealed increased protein plasticity, minor conformers and line broadening. The merit of fractional fluorine labeling in reducing such disruptions was demonstrated, and labeling levels of 60-75% provided an optimal balance between native-likeness and the usual advantages of 19F NMR in our system. The 19F NMR techniques developed here are broadly applicable and will expand the utility of 19F NMR in studies of protein systems.