No description available.

No description available.

Tyrosine kinases (TK) are important for cardiac function, but their downstream targets in the adult heart have yet to be established. The ShcA docking protein binds specific phosphotyrosine (pTyr) sites on activated TKs through its N-terminal PTB and C-terminal SH2 domains and stimulates downstream pathways through motifs such as pTyr sites in its central CH1 region. To explore the role of this TK scaffold in the adult heart, we generated a myocardial-specific knockout of murine ShcA (ShcA CKO). Such mice developed a dilated cardiomyopathy phenotype involving impaired systolic function with enhanced cardiomyocyte contractility. This uncoupling of global heart and intrinsic myocyte functions was associated with altered perimysial collagen and extracellular matrix complicance properties, suggesting disruption of mechanical coupling. In vivo dissection of ShcA signaling properties revealed that selective inactivation of the PTB domain in the myocardium had effects resembling those seen in ShcA CKO mice, while disruption of the SH2 domain caused a less severe cardiac phenotype. Downstream signaling through the CH1 pTyr sites was dispensable for baseline cardiac function, but necessary to prevent adverse remodeling after hemodynamic overload. Therefore, ShcA mediates pTyr signaling in the adult heart through multiple distinct signaling elements that control myocardial functions and response to stresses.

Tyrosine kinases (TK) are important for cardiac function, but their downstream targets in the adult heart have yet to be established. The ShcA docking protein binds specific phosphotyrosine (pTyr) sites on activated TKs through its N-terminal PTB and C-terminal SH2 domains and stimulates downstream pathways through motifs such as pTyr sites in its central CH1 region. To explore the role of this TK scaffold in the adult heart, we generated a myocardial-specific knockout of murine ShcA (ShcA CKO). Such mice developed a dilated cardiomyopathy phenotype involving impaired systolic function with enhanced cardiomyocyte contractility. This uncoupling of global heart and intrinsic myocyte functions was associated with altered perimysial collagen and extracellular matrix complicance properties, suggesting disruption of mechanical coupling. In vivo dissection of ShcA signaling properties revealed that selective inactivation of the PTB domain in the myocardium had effects resembling those seen in ShcA CKO mice, while disruption of the SH2 domain caused a less severe cardiac phenotype. Downstream signaling through the CH1 pTyr sites was dispensable for baseline cardiac function, but necessary to prevent adverse remodeling after hemodynamic overload. Therefore, ShcA mediates pTyr signaling in the adult heart through multiple distinct signaling elements that control myocardial functions and response to stresses.

Split-protein systems are invaluable tools used for the discovery and investigations of the complexities of protein functions and interactions. Split-protein systems rely on the non-covalent interactions of two fragments of a split protein to restore protein function. Because of this, they have the ability to restore protein functions post-translationally, thus allowing for quick and efficient responses to a milieu of cellular mechanisms. Despite this, split-protein systems have been largely limited as a reporting tool for protein-protein interactions. The recent discovery of inteins has the potential of broadening the scope of split-protein systems. Inteins are protein elements that possess the unique ability of post-translationally ligating protein fragments together with a native peptide bond, a process termed protein splicing. This allows split-proteins to reassemble in a more natural state. Exploiting this property and utilizing protein engineering techniques and methodologies, several approaches are described here for restoring and controlling split-protein functions using inteins. First, the protein splicing behaviour was demonstrated with the development of a simple in vitro visual fluorescence assay that relies on examining the subcellular localization of different fluorescent proteins. Inteins were then used to reassemble and restore function to artificially split genetically encoded Ca2+ indicators. Second, inteins were shown to be able to simultaneously restore protein function to two target proteins. The first target protein was restored through the normal protein splicing pathway while the second was restored through non-covalent interactions of the split-protein fragments. This is a previous unknown property of inteins. Lastly, an intein was engineered to respond to an external light-stimulus that triggered protein splicing to restore split-protein function. The photoactivatable intein, coupled with the versatility of light, allows exquisite control in both space and time for the restoration of protein function within cells. The modularity of the photoactivatable intein can be simply attached to a variety of split-proteins. This was demonstrated with the restoration of various split-protein functions.

Regulators of G-protein signaling (RGS) modulate G-protein coupled receptor (GPCR) activity in vascular smooth muscle cells (VSMCs). One such protein, RGS5, has been shown to have selective expression in VSMCs and pericytes, and can inhibit signaling from Gαq and Gαi subunits. Using an RGS5 knockout model, we assessed the functional effect of RGS5 in the constriction and dilation of resistance arterioles. Furthermore, we examined the intracellular lipid interaction of RGS proteins to identify the determinants regulating the biologic function of RGS5. Surprisingly, loss of RGS5 function in mesenteric arterioles had no effect on constriction and dilation of resistance arterioles. Cultured VSMCs showed increased basal ERK1/2 phosphorylation and increased VASP signaling in response to SNP treatment in RGS5KO VSMCs as compared to wild type controls, with no effect on cell proliferation. These data suggest RGS5 may integrate multiple intracellular pathways with competing effects on VSMC contraction.

Split-protein systems are invaluable tools used for the discovery and investigations of the complexities of protein functions and interactions. Split-protein systems rely on the non-covalent interactions of two fragments of a split protein to restore protein function. Because of this, they have the ability to restore protein functions post-translationally, thus allowing for quick and efficient responses to a milieu of cellular mechanisms. Despite this, split-protein systems have been largely limited as a reporting tool for protein-protein interactions. The recent discovery of inteins has the potential of broadening the scope of split-protein systems. Inteins are protein elements that possess the unique ability of post-translationally ligating protein fragments together with a native peptide bond, a process termed protein splicing. This allows split-proteins to reassemble in a more natural state. Exploiting this property and utilizing protein engineering techniques and methodologies, several approaches are described here for restoring and controlling split-protein functions using inteins. First, the protein splicing behaviour was demonstrated with the development of a simple in vitro visual fluorescence assay that relies on examining the subcellular localization of different fluorescent proteins. Inteins were then used to reassemble and restore function to artificially split genetically encoded Ca2+ indicators. Second, inteins were shown to be able to simultaneously restore protein function to two target proteins. The first target protein was restored through the normal protein splicing pathway while the second was restored through non-covalent interactions of the split-protein fragments. This is a previous unknown property of inteins. Lastly, an intein was engineered to respond to an external light-stimulus that triggered protein splicing to restore split-protein function. The photoactivatable intein, coupled with the versatility of light, allows exquisite control in both space and time for the restoration of protein function within cells. The modularity of the photoactivatable intein can be simply attached to a variety of split-proteins. This was demonstrated with the restoration of various split-protein functions.

Regulators of G-protein signaling (RGS) modulate G-protein coupled receptor (GPCR) activity in vascular smooth muscle cells (VSMCs). One such protein, RGS5, has been shown to have selective expression in VSMCs and pericytes, and can inhibit signaling from Gαq and Gαi subunits. Using an RGS5 knockout model, we assessed the functional effect of RGS5 in the constriction and dilation of resistance arterioles. Furthermore, we examined the intracellular lipid interaction of RGS proteins to identify the determinants regulating the biologic function of RGS5. Surprisingly, loss of RGS5 function in mesenteric arterioles had no effect on constriction and dilation of resistance arterioles. Cultured VSMCs showed increased basal ERK1/2 phosphorylation and increased VASP signaling in response to SNP treatment in RGS5KO VSMCs as compared to wild type controls, with no effect on cell proliferation. These data suggest RGS5 may integrate multiple intracellular pathways with competing effects on VSMC contraction.

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.

The three dimensional structure of a protein is important for its function. When misfolded, a protein may be rendered inactive or adapt a conformation that could be toxic. Studying protein folding requires an understanding of protein conformation. Traditionally, protein conformation has been studied using x-ray crystallography and nuclear magnetic resonance (NMR). X-ray crystallography is limited in the analysis of crystallized proteins and is computationally intensive. NMR deals with proteins in solution but reports only an average of conformation and the technique severely suffers from spectral overlapping due to the thousands of resonances of the protein. More recently, mass spectrometry has been employed not only to elucidate primary structures but also gather information on the three-dimensional conformation of proteins. In this study, a mass spectrometric-based approach is used to study the changes in conformation of cytochrome c and the green fluorescent protein when subjected to aqueous-organic solvent systems. The technique involved trypsin digestion and generation of peptide mass maps. For cytochrome c, the experiments were done with ethanol, methanol and acetonitrile to gain insights on naturation and denaturation. An apparent solvent effect to the rate of digestion and propensity for missed cleavages attributed to weakening of hydrophobic interactions and strengthening of intramolecular hydrogen bonding was observed. For the green fluorescent protein, sulfolane, a known supercharging agent, was used to gain insights on the effect of supercharging to protein conformation. Addition of 2.0% sulfolane shifted the charge state envelope of the protein towards lower m/z while adding lower amounts of sulfolane enhanced lower charge states while broadening the charge state envelope. The time course study showed different patterns of digestion dependent on solvent conditions implying changes in conformation. Furthermore, absorbance and fluorescence measurements suggested that addition of sulfolane protects the fluorophore from quenching. The activity of trypsin is not affected by addition of low amounts of sulfolane.