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Studies of the relationship of protein structure to regulation and catalysis in tyrosine hydroxylaseSura, Giri Raju 17 September 2007 (has links)
Tyrosine hydroxylase (TyrH) catalyzes the rate-limiting step in the synthesis of the catecholamine neurotransmitters dopamine, epinephrine, and norepinephrine. Phosphorylation of Ser40 of rat TyrH activates the enzyme by decreasing the affinity for catecholamines. In humans, there are four different TyrH isoforms with varying lengths for the regulatory domain. DOPA and dopamine binding studies were performed on the phosphorylated and unphosphorylated human isoforms. The Kd for DOPA was increased two times upon phosphorylation of hTyrH1, but no change was seen for hTyrH4; the Kd value decreased with the increase in the size of regulatory domain. The small effect on the Kd value for DOPA upon phosphorylation of hTyrH suggests that DOPA does not regulate the activity of hTyrH. Dopamine binds very tightly and upon phosphorylation the affinity for dopamine is decreased. This Kd value decreases with the increase in the length of the regulatory domain. The crystal structures of substrate complexes of the homologous enzyme phenylalanine hydroxylase (PheH) show a large movement of a surface loop (residues 131-155) upon amino acid binding. The corresponding loop residues (175-200) in TyrH play an important role in DOPA formation. This conformational change in TyrH loop was studied with fluorescence anisotropy. Three tryptophan residues in the TyrH, at positions 166, 233, and 372, were mutated to phenylalanine, and Phe184 was mutated to tryptophan. An increase in anisotropy was observed in the presence of phenylalanine and 6-methyl-5-deazatetrahydropterin (6M5DPH4), but the magnitude of the change of anisotropy with 6M5DPH4 was greater than that with phenylalanine. Further characterization of the sole tryptophan in the loop showed a decrease in the amplitude of the local motion only in the presence of 6M5DPH4 alone. The conformational change in wild type TyrH was examined by H/D exchange LC/MS spectroscopy in the presence of the natural ligands. Time-course dependent deuterium incorporation into the loop in the presence of ligands indicated that the pterin alone can induce the conformational change in the loop irrespective of whether iron is reduced or oxidized. From these results, one can conclude that the loop undergoes a conformational change upon pterin binding, making the active site better for amino acid binding.
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Studies of the relationship of protein structure to regulation and catalysis in tyrosine hydroxylaseSura, Giri Raju 17 September 2007 (has links)
Tyrosine hydroxylase (TyrH) catalyzes the rate-limiting step in the synthesis of the catecholamine neurotransmitters dopamine, epinephrine, and norepinephrine. Phosphorylation of Ser40 of rat TyrH activates the enzyme by decreasing the affinity for catecholamines. In humans, there are four different TyrH isoforms with varying lengths for the regulatory domain. DOPA and dopamine binding studies were performed on the phosphorylated and unphosphorylated human isoforms. The Kd for DOPA was increased two times upon phosphorylation of hTyrH1, but no change was seen for hTyrH4; the Kd value decreased with the increase in the size of regulatory domain. The small effect on the Kd value for DOPA upon phosphorylation of hTyrH suggests that DOPA does not regulate the activity of hTyrH. Dopamine binds very tightly and upon phosphorylation the affinity for dopamine is decreased. This Kd value decreases with the increase in the length of the regulatory domain. The crystal structures of substrate complexes of the homologous enzyme phenylalanine hydroxylase (PheH) show a large movement of a surface loop (residues 131-155) upon amino acid binding. The corresponding loop residues (175-200) in TyrH play an important role in DOPA formation. This conformational change in TyrH loop was studied with fluorescence anisotropy. Three tryptophan residues in the TyrH, at positions 166, 233, and 372, were mutated to phenylalanine, and Phe184 was mutated to tryptophan. An increase in anisotropy was observed in the presence of phenylalanine and 6-methyl-5-deazatetrahydropterin (6M5DPH4), but the magnitude of the change of anisotropy with 6M5DPH4 was greater than that with phenylalanine. Further characterization of the sole tryptophan in the loop showed a decrease in the amplitude of the local motion only in the presence of 6M5DPH4 alone. The conformational change in wild type TyrH was examined by H/D exchange LC/MS spectroscopy in the presence of the natural ligands. Time-course dependent deuterium incorporation into the loop in the presence of ligands indicated that the pterin alone can induce the conformational change in the loop irrespective of whether iron is reduced or oxidized. From these results, one can conclude that the loop undergoes a conformational change upon pterin binding, making the active site better for amino acid binding.
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Development and implementation of a FT-ICR mass spectrometer for the investigation of ion conformations of peptide sequence isomers containing basic amino acid residues by gas-phase hydrogen/deuterium exchangeMarini, Joseph Thomas 30 September 2004 (has links)
The gas-phase hydrogen/deuterium (H/D) exchange of protonated di- and tripeptides containing a basic amino acid residue has been studied with a Fourier transform ion cyclotron resonance (FT-ICR) mass spectrometer. Bimolecular reactions are monitored as a function of time providing exchange efficiencies and temporal distributions for the peptide ions. Results from these experiments indicated that position of the basic residue within the peptide (i.e. N-terminal, internal, or C-terminal) influences gas-phase H/D exchange, suggesting unique peptide ion conformations. The FT-ICR mass spectrometer employed for these gas-phase H/D exchange studies was modified from its original design. Instrument modifications include development of an internal matrix assisted laser desorption ionization (MALDI) source for peptide protonation. In addition, a two-section cell was utilized, allowing control of ion motion and factors affecting gas-phase ion molecule reactions. Systems investigated in these gas-phase H/D exchange studies are peptides containing the same amino acid residues but different sequences. These sequence isomers display dissimilar reaction efficiencies and temporal distributions for deuterium incorporation depending on the primary structure of the peptide ion. Specifically, [M+H]+ peptide ions containing a N-terminal basic residue demonstrate unique H/D exchange behavior when compared to their internal and C-terminal counterparts. These differences are attributed to dissimilar intramolecular bridging interactions involved with inductive stabilization of the charge site. Gas-phase H/D exchange of peptide sequence isomers was also probed with various deuterium reagents. Findings suggest that different reagents also influence H/D exchange reaction rate efficiencies and temporal distributions. These dissimilarities are ascribed to relative gas-phase basicity and proposed mechanistic exchange differences for the deuterium reagents.
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Mass Spectrometry-Based Strategies for Multiplexed Analyses of Protein-Ligand Binding InteractionsDeArmond, Patrick D. January 2011 (has links)
<p>The detection and quantitation of protein-ligand binding interactions is important not only for understanding biological functions but also for the characterization of novel protein ligands. Because protein ligands can range from small molecules to other proteins, general techniques that can detect and quantitate the many classes of protein-ligand interactions are especially attractive. Additionally, the ability to detect and quantify protein-ligand interactions in complex biological mixtures would more accurately represent the protein-ligand interactions that occur in vivo, where differential protein expression and protein complexes can significantly affect a protein's ability to bind to a ligand of interest.</p><p> The work in this dissertation is focused on the development of new methodologies for the detection and measurement of protein-ligand interactions in complex mixtures using multiplex analyses. Methodologies for two types of multiplexed analyses of protein-ligand binding interactions are investigated here. The first type of multiplex analysis involves characterizing the binding of one protein target to many potential ligands, and the second type involves characterizing the binding of one ligand to many proteins. The described methodologies are derived from the SUPREX (stability of unpurified proteins from rates of H/D exchange) and SPROX (stability of proteins from rates of oxidation) techniques, which are chemical modification strategies that measure thermodynamic stabilities of proteins using a relationship between a protein's folding equilibrium and the extent of chemical modification. These two techniques were utilized in the development and application of several different experimental strategies designed to multiplex the analysis of protein-ligand interactions.</p><p> The first strategy that was developed involved a pooled compound approach for making SUPREX-based measurements of multiple ligands binding to a target protein. Screening rates of 6 s/ligand were demonstrated in a high-throughput screening project that involved the screening of two chemical libraries against human cyclophilin A (CypA), a protein commonly overexpressed in types of cancer. This study identified eight novel ligands to CypA with micromolar dissociation constants. Second, an affinity-based protein purification strategy was developed for the detection and quantitation of specific protein-ligand binding interactions in the context of complex protein mixtures. It involved performing SPROX in cell lysates and selecting the protein of interest using immunoprecipitation or affinity tag purification. A third strategy developed here involved a SPROX-based stable isotope labeling method for measuring protein-ligand interactions in multi-protein mixtures. This strategy was used in a proof-of-principle experiment designed to detect and quantify the indirect binding between yeast cyclophilin and calcineurin in a multi-component protein mixture. Finally, a quantitative proteomics platform was developed for the detection and quantitation of protein-ligand binding interactions on the proteomic scale. The platform was used to profile interactions of the proteins in a yeast cell lysate to several ligands, including the bioactive small molecules resveratrol and manassantin A, the cofactor nicotinamide adenine dinucleotide (NAD+), and two proteins, phosphoglycerate kinase (Pgk1) and pyruvate kinase (Pyk1). The above approaches should have broad application for use as discovery tools in the development of new therapeutic agents.</p> / Dissertation
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Explorations of iron-iron hydrogenase active site models by experiment and theoryTye, Jesse Wayne 15 May 2009 (has links)
This dissertation describes computational and experimental studies of synthetic
complexes that model the active site of the iron-iron hydrogenase [FeFe]H2ase enzyme.
Simple dinuclear iron dithiolate complexes act as functional models of the ironiron
hydrogenase enzyme by catalyzing isotopic exchange in D2/H2O mixtures. Density
Functional Theory (DFT) calculations and new experiments have been performed that
suggest reasonable mechanistic explanations for this reactivity. Evidence for the
existence of an acetone derivative of the di-iron complex, as suggested by theory, is
presented.
Bis-phosphine substituted dinuclear iron dithiolate complexes react with the
electrophilic species, H+ and Et+ (Et+ = CH3CH2
+) with differing regioselectivity; H+
reacts to form a 3c-2eâ Fe-H-Fe bond, while Et+ reacts to form a new C-S bond. The
instability of a bridging ethyl complex is attributed to the inability of the ethyl group, in
contrast to a hydride, to form a stable 3c-2eâ bond with the two iron centers.
Gas-phase density functional theory calculations are used to predict the solutionphase
infrared spectra for a series of CO and CN-containing dinuclear iron complexes
dithiolate. It is shown that simple linear scaling of the computed C-O and C-N stretching frequencies yields accurate predictions of the experimentally determined ν(CO) and
ν(CN) values.
An N-heterocyclic carbene containing [FeFe]H2ase model complex, whose X-ray
structure displays an apical carbene, is shown to undergo an unexpected simultaneous
two-electron reduction. DFT shows, in addition to a one-electron Fe-Fe reduction, that
the aryl-substituted N-heterocyclic carbene can accept a second electron more readily
than the Fe-Fe manifold. The juxtaposition of these two one-electron reductions
resembles the [FeFe]H2ase active site with an FeFe di-iron unit joined to the
electroactive 4Fe4S cluster.
Simple synthetic di-iron dithiolate complexes synthesized to date fail to
reproduce the precise orientation of the diatomic ligands about the iron centers that is
observed in the molecular structure of the reduced form of the enzyme active site.
Herein, DFT computations are used for the rational design of synthetic complexes as
accurate structural models of the reduced form of the enzyme active site.
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Millisecond H/D Exchange Combined with Electrospray Ionization Mass Spectrometry to Study Protein¡¦s StructureLin, Hsuan-Chung 03 August 2004 (has links)
none
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Thermal Chemistry of 2-Propynyl Bromide and 1-Propynyl Iodide on the Ag(111) SurfaceWu, Yu-Jui 19 July 2001 (has links)
none
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Millisecond H/D Exchange Combined with Electrospray Ionization Mass Spectrometry to Study Three Dimensional Structure of ProteinHuang, Ming-Wei 23 June 2003 (has links)
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Investigation of the effect of intra-molecular interactions on the gas-phase conformation of peptides as probed by ion mobility-mass spectrometry, gas-phase hydrogen/deuterium exchange, and molecular mechanicsSawyer, Holly Ann 12 April 2006 (has links)
Ion mobility-mass spectrometry (IM-MS), gas-phase hydrogen/deuterium (H/D) exchange ion molecule reactions and molecular modeling provide complimentary information and are used here for the characterization of peptide ion structure, including fine structure detail (i.e., cation-π interactions, β-turns, and charge solvation interactions). IM-MS experiments performed on tyrosine containing tripeptides show that the collision cross-sections of sodiated, potassiated and doubly sodiated species of gly-gly-tyr are smaller than that of the protonated species, while the cesiated and doubly cesiated species are larger. Conversely, all of the alkali-adducted species of try-gly-gly have collision cross-sections that are larger than that of the protonated species. The protonated and alkali metal ion adducted (Na+, K+ and Cs+) species of bradykinin and bradykinin fragments 1-5, 1-6, 1-7, 1-8, 2-7, 5-9 and 2-9 were also studied using IM-MS and the alkali metal ion adducts of these species were found to have cross-sections very close to those of the protonated species. Additionally, multiple peak features observed in the ATDs of protonated bradykinin fragments 1-5, 1-6 and 1-7 are conserved upon alkali metal ion adduction. It was observed from gas-phase H/D ion molecule reactions that alkali adducted species exchange slower and to a lesser extent than protonated species in the tyrosine- and arginine-containing peptides. Experimental and computational results are discussed in terms of peptide ion structure, specifically the intra-molecular interactions present how those interactions change upon alkali salt adduction, as well as with the sequence of the peptide.
Additionally, IM-MS data suggests the presence of a compact conformation of bradykinin fragment 1-5 (RPPGF) when starting from organic solvent conditions. As water is added stepwise to methanolic solutions, a more extended conformation is populated. When the starting solution is composed of ≈90% water, two distinct mobility profiles are observed as well as a shoulder, indicating the presence of three gas-phase conformations for RPPGF. Gas-phase H/D exchange of [M+H]+ ions prepared from aqueous solvents show a bi-exponential decay, whereas samples prepared from organic solvents show a single exponential decay. The effect of solvent on gas-phase peptide ion structure, i.e., solution-phase memory effects, is discussed and gas-phase structures are compared to know solution-phase structures.
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PROBING GAS-PHASE PEPTIDE STRUCTURE AND PROTEIN-PROTEIN INTERACTIONS USING MASS SPECTROMETRIC TECHNIQUESPerkins, Brittany January 2009 (has links)
Presented in this dissertation are studies on the gas-phase structural features of peptides and peptide fragment ions using mass spectrometry (MS), hydrogen/deuterium (H/D) exchange, infrared multiphoton dissociation (IRMPD) spectroscopy, and computational modeling. Additional studies are presented on the mechanism of hydrogen/deuterium exchange using a model amino acid system. The application of chemical cross-linking to investigate the interaction between two proteins, LexA and RecA, is also presented. Gas-phase structural features can be probed using a number of techniques, and several of the studies presented in this dissertation involve the use of gas-phase H/D exchange. Although the basic mechanism for exchange has been determined, the factors that affect the rate and extent of exchange are not well understood. A computational modeling study of the exchange behavior of asparagine and its methyl ester demonstrated that exchange will occur preferentially at sites of more similar basicity. The distinctive exchange behavior of a model histidine-containing pentapeptide, HAAAA, prompted further studies into the structural features that result in five fast exchanging hydrogens and one slower exchange. Peptide analogues were used to identify the sites of exchange, and IRMPD spectroscopy combined with computational modeling indicated that exchange may occur because interaction with water at those sites results in lower energy structures compared to the other sites. Structural studies were also performed to determine whether the b₂⁺ ion from HAAAA is an oxazolone or diketopiperazine. Although the IRMPD spectrum matched that of a diketopiperazine, H/D exchange and fragmentation studies revealed the presence of both a diketopiperazine and oxazolone structure. Protein-protein interactions perform a vital role in regulating cellular processes. Despite extensive mutational analysis, the binding interaction between LexA and RecA, two proteins involved in the SOS response, is unclear. Chemical cross-linking experiments were undertaken to help target future mutational studies, and these studies identified two possible interactions. The first potential binding interaction is located in the cleft of RecA, and the second interaction may be caused by a LexA dimer binding across the RecA helical groove. The presence of two different binding interactions suggests that LexA may have redundant binding modes for RecA interaction.
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