Global ETD Search

1	Understanding Ultrafast Hydration Dynamics under Crowding Condition and Tryptophan Fluorescence Quenching Mechanism in Gamma-M7 Crystallin Yang, Yushan January 2021 (has links) No description available. Physics Biophysics
2	Palmitoylation and Oxidation of the Cysteine Rich Region of SNAP-25 and their Effects on Protein Interactions Martinez, Derek Luberli 17 July 2007 (has links) (PDF) Neurons depend upon neurotransmitter release through regulated exocytosis to accomplish the immense processing performed within the central nervous system. The SNARE hypothesis points to a family of proteins that are thought to enable the membrane fusion that leads to exocytosis. The secondary structure of SNAP-25 is unique among SNARE proteins in that it has two alpha helical SNARE motifs and a cysteine rich (C85, C88, C90, C92) membrane interacting region but notransmembrane domain. The cysteines may be modified by palmitoylation or oxidation but the role of these modifications in vivo is not well understood. Our goal is to elucidate possible regulatory roles of SNAP-25 that relate to its unique structure and these reversible modifications. However, the study of SNAP-25 in reconstituted systems is hampered by a lack of readily available palmitoylated SNAP-25. A method for in vitro palmitoylation of SNAP-25 by HIP14, a neuronal acyltransferase, is described along with the application of a biotinylation streptavidin assay to verify palmitoylation. Palmitoylation increases the extent to which SNAP-25 interacts with lipids as observed with an environment sensitive trpytophan fluorescence assay. Palmitoylation also alters the phase transition of DPPC lipids differently than unpalmitoylated SNAP-25.This effect on the membrane may influence fusion events. Oxidation of the cysteine residues may be responsible for the sensitivity of SNAP-25 to reactive oxygen species. Our data suggests that, when oxidized, SNAP-25 does not interact with membranes to the same extent as palmitoylated SNAP-25. This may provide a mechanism for reducing exocytosis during oxidative stress. Also, oxidized SNAP-25 is not susceptible to Botulinum Neurotoxin E. The effects of oxidation and palmitoylation on the protein interactions of SNAP-25 may shed light on its role in the SNARE complex and membrane fusion. SNAP-25 palmitoylation SNARE N-Ethylmaleimide biotin exocytosis membrane fusion Botulinum Neurotoxin differential scanning calorimetry tryptophan fluorescence oxidation Neuroscience and Neurobiology
3	Membrane binding properties of Disabled-2 Alajlouni, Ruba 10 May 2011 (has links) Disabled-2 (Dab2) is an adapter protein that interacts with cell membranes and it is involved in several biological processes including endocytosis and platelet aggregation. During endocytosis, the Dab2 phosphotyrosine-binding (PTB) domain mediates protein binding to phosphatidylinositol 4,5-bisphosphate (PIP2) at the inner leaflet of the plasma membrane and helps co-localization with clathrin coats. Dab2, released from platelet alpha granules, inhibits platelet aggregation by binding to the °IIb? integrin receptor on the platelet surface through an Arg-Gly-Asp (RGD) motif located within the PTB domain. Alternatively, Dab2 binds sulfatides on the platelets surface, and this binding partition Dab2 in two pools (sulfatide and integrin receptor-bound states), but the biological consequences of lipid binding remain unclear. Dab2 binds sulfatides through two basic motifs located on its N-terminal region including the PTB domain (N-PTB). We have characterized the binding of Dab2 to micelles, which are widely used to mimic biological membranes. These micellar interactions were studied in the absence and presence of Dab2 lipid ligands, sulfatides and PIP2. By applying multiple biochemical, biophysical, and structural techniques, we found that whereas Dab2 N-PTB binding to PIP2 stabilized the protein but did not contribute to the penetration of the protein into micelles, sulfatides induced conformational changes and facilitated penetration of Dab2 N-PTB into micelles. This is in agreement with previous observation that sulfatides, but not PIP2, protect Dab2 N-PTB from thrombin cleavage. By studying the mechanism by which Dab2 targets membranes, we will have the opportunity to manipulate its function in different lipid-dependent biological processes. / Master of Science 5-bisphosphate Circular Dichroism Micelles Sulfatide Tryptophan Fluorescence Disabled-2 Nuclear Magnetic Resonance Phosphatidylinositol 4 Acrylamide quenching
4	Dinâmica Molecular de Peptídeos na Interface Membrana-Água / Molecular dynamics of peptides in the membrane-water interface Pascutti, Pedro Geraldo 25 October 1996 (has links) Um programa computacional foi desenvolvido para otimização de geometria e simulação de dinâmica molecular baseado em um campo de forças clássicas parametrizado. O solvente foi considerado como um contínuo eletrostático e a interface entre o meio aquoso e o interior de uma membrana biológica como uma superfície de descontinuidade dielétrica, tratada pelo \"método das imagens eletrostáticas\". Nesse método, o campo de polarização produzido na superfície de descontinuidade por uma carga pontual é representado por uma carga fictícia, colocada na fase oposta, cuja distância e sinal é definida pelas condições de contorno na superfície. Diversos sistemas foram estudados, tanto em solventes contínuos como na presença de superfícies de descontinuidade: a) Foram estudadas as distribuições populacionais dos rotâmeros do triptofano na forma zwitteriônica e no peptídeo Ala-Trp- Ala, em solvente polar e apolar. Foi demonstrado que a dinâmica do triptofano e as populações de rotâmeros são compatíveis com as observações experimentais de fluorescência resolvida no tempo e NMR; b) Em um estudo das conformações em polialanina, verificou-se que a estabilidade da estrutura secundária hélice- é um efeito cooperativo entre pontes de hidrogênio em solvente de baixa constante dielétrica. Na presença da interface água-membrana, a hélice- anfifílica de um modelo para a -endorfina estabiliza-se sobre a interface. Um comportamento anfifílico foi também observado na seqüência sinal para o receptor- da e. coli, a qual estabilizou-se perpendicularmente à interface, na conformação parcial hélice- proposta na literatura; c) Em um estudo sobre o hormônio -MSH observou-se que, em solvente polar, de uma conformação helicoidal ele passa para uma conformação estendida. Porém, ao atravessar para o interior hidrofóbico de uma membrana, o peptídeo estabiliza-se em dobra-. Observou-se ainda que a estabilidade dessa conformação no interior da membrana é reforçada por pontes salinas entre os resíduos carregados do peptídeo, os quais formam um \"caroço\" hidrofílico circundado por resíduos hidrofóbicos. Esse arranjo estrutural está em concordância com o proposto para a conformação biologicamente ativa. De um modo geral, o modelo para biomembrana proposto no presente trabalho reproduziu o comportamento hidrofóbico, hidrofílico ou anfifílico dos peptídeos estudados. / A software was developed for optimisation of geometry and molecular dynamics simulation, based on a parameterized classical force field. Solvent was assumed as an electrostatic continuum. The interface between the aqueous medium and the hydrophobic core of biological membranes was described by a surface of dielectric discontinuity, treated by the \"method of images\". In this method, the polarization field produced at the surface of discontinuity by a point charge was represented by a fictitious charge, placed in the opposite phase. The position and signal of this charge-image were defined by boundary conditions at the surface. Several systems were studied, either in continuous solvent, as in the presence of discontinuity surfaces: a) the population distribution of tryptophan rotamers was studied in the zwytterion and in the peptide Ala-Trp-Ala, in polar and apolar solvents; the results for the tryptophan dynamics and the rotamers populations agree with experimental observations using time resolved fluorescence and NMR spectroscopies. b) analysis of polyalanin conformations showed that the stabililty of the -helix is a cooperative effect between hydrogen bonds in low dielectric constant solvent; in the presence of the water-membrane interface, the amphyphilic -helix of a -endorphin model stabilizes on the interface; a similar behavior was observed in the signal sequence for the E. Coli -receptor, that stabilized perpendicular to the interface in a partial -helix conformation, as proposed in the literature. c) calculations on melanotropic hormone a.-MSH showed that in polar solvent it goes from helycoidal conformation to an extended one; in the presence of the interface water-membrane, the peptide goes into the interior of the membrane and stabilizes in a -turn; the stability ofthis conformation was reinforced by salt bridges between charged residues, forming a hydrophilic core surrounded by hydrophobic residues; this structural arrangement agrees with the one proposed for the biologically active conformation of the hormone. In general terms, the model proposed here for the biomembrane was able to mimic the hydrophobic, hydrophihlic or amphyphilic behavior of the peptides studied. Cell membranes Dinâmica molecular Electrostatic images Estruturas secundárias Fluorescence Fluorescência Fluorescência do triptofano Imagens eletrostáticas Membranas celulares Molecular dynamics Peptídeos Peptides Secondary structures Tryptophan fluorescence
5	Dinâmica Molecular de Peptídeos na Interface Membrana-Água / Molecular dynamics of peptides in the membrane-water interface Pedro Geraldo Pascutti 25 October 1996 (has links) Um programa computacional foi desenvolvido para otimização de geometria e simulação de dinâmica molecular baseado em um campo de forças clássicas parametrizado. O solvente foi considerado como um contínuo eletrostático e a interface entre o meio aquoso e o interior de uma membrana biológica como uma superfície de descontinuidade dielétrica, tratada pelo \"método das imagens eletrostáticas\". Nesse método, o campo de polarização produzido na superfície de descontinuidade por uma carga pontual é representado por uma carga fictícia, colocada na fase oposta, cuja distância e sinal é definida pelas condições de contorno na superfície. Diversos sistemas foram estudados, tanto em solventes contínuos como na presença de superfícies de descontinuidade: a) Foram estudadas as distribuições populacionais dos rotâmeros do triptofano na forma zwitteriônica e no peptídeo Ala-Trp- Ala, em solvente polar e apolar. Foi demonstrado que a dinâmica do triptofano e as populações de rotâmeros são compatíveis com as observações experimentais de fluorescência resolvida no tempo e NMR; b) Em um estudo das conformações em polialanina, verificou-se que a estabilidade da estrutura secundária hélice- é um efeito cooperativo entre pontes de hidrogênio em solvente de baixa constante dielétrica. Na presença da interface água-membrana, a hélice- anfifílica de um modelo para a -endorfina estabiliza-se sobre a interface. Um comportamento anfifílico foi também observado na seqüência sinal para o receptor- da e. coli, a qual estabilizou-se perpendicularmente à interface, na conformação parcial hélice- proposta na literatura; c) Em um estudo sobre o hormônio -MSH observou-se que, em solvente polar, de uma conformação helicoidal ele passa para uma conformação estendida. Porém, ao atravessar para o interior hidrofóbico de uma membrana, o peptídeo estabiliza-se em dobra-. Observou-se ainda que a estabilidade dessa conformação no interior da membrana é reforçada por pontes salinas entre os resíduos carregados do peptídeo, os quais formam um \"caroço\" hidrofílico circundado por resíduos hidrofóbicos. Esse arranjo estrutural está em concordância com o proposto para a conformação biologicamente ativa. De um modo geral, o modelo para biomembrana proposto no presente trabalho reproduziu o comportamento hidrofóbico, hidrofílico ou anfifílico dos peptídeos estudados. / A software was developed for optimisation of geometry and molecular dynamics simulation, based on a parameterized classical force field. Solvent was assumed as an electrostatic continuum. The interface between the aqueous medium and the hydrophobic core of biological membranes was described by a surface of dielectric discontinuity, treated by the \"method of images\". In this method, the polarization field produced at the surface of discontinuity by a point charge was represented by a fictitious charge, placed in the opposite phase. The position and signal of this charge-image were defined by boundary conditions at the surface. Several systems were studied, either in continuous solvent, as in the presence of discontinuity surfaces: a) the population distribution of tryptophan rotamers was studied in the zwytterion and in the peptide Ala-Trp-Ala, in polar and apolar solvents; the results for the tryptophan dynamics and the rotamers populations agree with experimental observations using time resolved fluorescence and NMR spectroscopies. b) analysis of polyalanin conformations showed that the stabililty of the -helix is a cooperative effect between hydrogen bonds in low dielectric constant solvent; in the presence of the water-membrane interface, the amphyphilic -helix of a -endorphin model stabilizes on the interface; a similar behavior was observed in the signal sequence for the E. Coli -receptor, that stabilized perpendicular to the interface in a partial -helix conformation, as proposed in the literature. c) calculations on melanotropic hormone a.-MSH showed that in polar solvent it goes from helycoidal conformation to an extended one; in the presence of the interface water-membrane, the peptide goes into the interior of the membrane and stabilizes in a -turn; the stability ofthis conformation was reinforced by salt bridges between charged residues, forming a hydrophilic core surrounded by hydrophobic residues; this structural arrangement agrees with the one proposed for the biologically active conformation of the hormone. In general terms, the model proposed here for the biomembrane was able to mimic the hydrophobic, hydrophihlic or amphyphilic behavior of the peptides studied. Dinâmica molecular Estruturas secundárias Fluorescência Fluorescência do triptofano Imagens eletrostáticas Membranas celulares Peptídeos Cell membranes Electrostatic images Fluorescence Molecular dynamics Peptides Secondary structures Tryptophan fluorescence
6	Characterization of the Interactions Mediated by the Key Structural Protein CcmL: Cornerpiece of the Beta-Carboxysome Keeling, Thomas 16 January 2013 (has links) While much is known about the structure and interactions of the β-carboxysomal proteins, interactions of the proposed vertex protein CcmL with the other components have not yet been directly demonstrated. A fluorescence resonance energy transfer based method combined with other complementary spectrophotometry techniques as well as x-ray crystallography and transmission electron microscopy was used in a Thermosynechococcus elongatus BP-1 model to study these interactions. CcmL was found to interact with the various CcmK shell proteins with a clear binding preference for CcmK2; the previously proposed interaction of CcmL with CcmM was shown to not occur in vitro, and a possible CcmL-CcmL interaction was observed tentatively. In addition, analysis of a novel x-ray crystal structure of Nostoc sp. PCC7120 CcmL in a decameric form suggests a possibility of a CcmL-CcmL back-to-back interaction. This study gives the first direct experimental evidence for the biological role of CcmL as the carboxysomal vertex protein. carboxysome protein cyanobacteria fluorescence electron microscopy FRET x-ray crystallography protein interaction tryptophan fluorescence bacterial microcompartment dynamic light scattering Thermosynechococcus elongatus Nostoc sp. PCC7120 fluorescence resonance energy transfer
7	Fluorescence Detectors for Proteins and Toxic Heavy Metals Paul, Uchenna Prince 21 April 2004 (has links) (PDF) An inexpensive detector for proteins is described. The detection technique was based on two-photon excitation intrinsic protein fluorescence using a visible 532 nm diode-pumped nano laser as the excitation source. Proteins that exhibit intrinsic fluorescence must contain at least one tryptophan, tyrosine, or phenylalanine residue in their amino acid sequences. The detector was characterized and was found to have a detection limit of 4 micro-molar for tryptophan, 22 micro-molar for tyrosine and 500 micro-molar for phenylalanine. Bovine serum albumin, a serum protein with 3 tryptophan residues in its amino acid sequence was also used to characterize the detector. It was found that the detection limit for this protein was 0.9 micro-molar. The detector volume was determined based on a photon counting histogram - a technique in fluorescence fluctuation spectroscopy. From the results of this analysis, the excitation volume was found to be 2.9 fL. With such an excitation volume, the detection limits were either within or below the atto-mole range. protein detection intrinsic protein fluorescence tryptophan fluorescence tyrosine fluorescence excitation volume chemosensors cadmium detection Biochemistry Chemistry
8	Structural Investigation of Processing α-Glucosidase I from Saccharomyces cerevisiae Barker, Megan 20 August 2012 (has links) N-glycosylation is the most common eukaryotic post-translational modification, impacting on protein stability, folding, and protein-protein interactions. More broadly, N-glycans play biological roles in reaction kinetics modulation, intracellular protein trafficking, and cell-cell communications. The machinery responsible for the initial stages of N-glycan assembly and processing is found on the membrane of the endoplasmic reticulum. Following N-glycan transfer to a nascent glycoprotein, the enzyme Processing α-Glucosidase I (GluI) catalyzes the selective removal of the terminal glucose residue. GluI is a highly substrate-specific enzyme, requiring a minimum glucotriose for catalysis; this glycan is uniquely found in biology in this pathway. The structural basis of the high substrate selectivity and the details of the mechanism of hydrolysis of this reaction have not been characterized. Understanding the structural foundation of this unique relationship forms the major aim of this work. To approach this goal, the S. cerevisiae homolog soluble protein, Cwht1p, was investigated. Cwht1p was expressed and purified in the methyltrophic yeast P. pastoris, improving protein yield to be sufficient for crystallization screens. From Cwht1p crystals, the structure was solved using mercury SAD phasing at a resolution of 2 Å, and two catalytic residues were proposed based upon structural similarity with characterized enzymes. Subsequently, computational methods using a glucotriose ligand were applied to predict the mode of substrate binding. From these results, a proposed model of substrate binding has been formulated, which may be conserved in eukaryotic GluI homologs. Biomolecular structure Eukaryotic glycosylation glycosylation N-glycan N-linked glycosylation Carbohydrate biochemistry Inhibitor antiviral therapeutic Structure-based drug design glucosidase glycosidase glycoside hydrolase post-translational modification enzyme enzyme mechanism inverting mechanism structural biology protein structure 6xHis tag Crystal packing sugar substrate X-ray crystallography single anomalous dispersion PHENIX heavy-atom phasing docking autodock autodock vina biophysical methods tryptophan fluorescence glucose oxidase activity assay enzyme inhibition Protein expression Protein purification Pichia pastoris Saccharomyces cerevisiae fondue AOX1 promoter glycoside hydrolysis substrate binding model molecular dynamics molecular dynamics Binding site Active site cleft Endoplasmic reticulum Glycan processing Glycan trimming protein-protein interactions cell-cell communication X-ray diffraction crystallization secreted protein substrate selectivity kojibiose glucotriose miglitol deoxynojirimycin Glc3Man9GlcNAc2 free oligosaccharide species GluI Cwh41 Cwh41p Cwht1p 0306 0307 0760
9	Structural Investigation of Processing α-Glucosidase I from Saccharomyces cerevisiae Barker, Megan 20 August 2012 (has links) N-glycosylation is the most common eukaryotic post-translational modification, impacting on protein stability, folding, and protein-protein interactions. More broadly, N-glycans play biological roles in reaction kinetics modulation, intracellular protein trafficking, and cell-cell communications. The machinery responsible for the initial stages of N-glycan assembly and processing is found on the membrane of the endoplasmic reticulum. Following N-glycan transfer to a nascent glycoprotein, the enzyme Processing α-Glucosidase I (GluI) catalyzes the selective removal of the terminal glucose residue. GluI is a highly substrate-specific enzyme, requiring a minimum glucotriose for catalysis; this glycan is uniquely found in biology in this pathway. The structural basis of the high substrate selectivity and the details of the mechanism of hydrolysis of this reaction have not been characterized. Understanding the structural foundation of this unique relationship forms the major aim of this work. To approach this goal, the S. cerevisiae homolog soluble protein, Cwht1p, was investigated. Cwht1p was expressed and purified in the methyltrophic yeast P. pastoris, improving protein yield to be sufficient for crystallization screens. From Cwht1p crystals, the structure was solved using mercury SAD phasing at a resolution of 2 Å, and two catalytic residues were proposed based upon structural similarity with characterized enzymes. Subsequently, computational methods using a glucotriose ligand were applied to predict the mode of substrate binding. From these results, a proposed model of substrate binding has been formulated, which may be conserved in eukaryotic GluI homologs. Biomolecular structure Eukaryotic glycosylation glycosylation N-glycan N-linked glycosylation Carbohydrate biochemistry Inhibitor antiviral therapeutic Structure-based drug design glucosidase glycosidase glycoside hydrolase post-translational modification enzyme enzyme mechanism inverting mechanism structural biology protein structure 6xHis tag Crystal packing sugar substrate X-ray crystallography single anomalous dispersion PHENIX heavy-atom phasing docking autodock autodock vina biophysical methods tryptophan fluorescence glucose oxidase activity assay enzyme inhibition Protein expression Protein purification Pichia pastoris Saccharomyces cerevisiae fondue AOX1 promoter glycoside hydrolysis substrate binding model molecular dynamics molecular dynamics Binding site Active site cleft Endoplasmic reticulum Glycan processing Glycan trimming protein-protein interactions cell-cell communication X-ray diffraction crystallization secreted protein substrate selectivity kojibiose glucotriose miglitol deoxynojirimycin Glc3Man9GlcNAc2 free oligosaccharide species GluI Cwh41 Cwh41p Cwht1p 0306 0307 0760

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