Academic literature on the topic 'Intrinsic protein fluorescence'
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Journal articles on the topic "Intrinsic protein fluorescence"
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Goldberg, Jacob M., Rebecca F. Wissner, Alyssa M. Klein, and E. James Petersson. "Thioamide quenching of intrinsic protein fluorescence." Chemical Communications 48, no. 10 (2012): 1550–52. http://dx.doi.org/10.1039/c1cc14708k.
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Brewis, Neil, Anne Phelan, Jeanette Webb, Jeff Drew, Gill Elliott, and Peter O'Hare. "Evaluation of VP22 Spread in Tissue Culture." Journal of Virology 74, no. 2 (January 15, 2000): 1051–56. http://dx.doi.org/10.1128/jvi.74.2.1051-1056.2000.
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ABSTRACT We compare methods of detection of intercellular transport of the herpes simplex virus protein VP22 and of a green fluorescent protein (GFP)-VP22 fusion protein. Spread of both proteins was observed by immunofluorescence (IF) using organic fixatives. Spread of both proteins was also detected by IF after paraformaldehyde (PFA) fixation and detergent permeabilization, albeit at reduced levels. However, while spread of GFP-VP22 was observed by examining intrinsic GFP fluorescence after methanol fixation, little spread was observed after PFA fixation, suggesting that the levels of the fusion protein in recipient cells were below the detection limits of intrinsic-fluorescence or that PFA fixation quenches the fluorescence of GFP-VP22. We further considered whether elution of VP22 from methanol-fixed cells and postfixation binding to surrounding cells contributed to the increased detection of spread observed after methanol fixation. The results show that while this could occur, it appeared to be a minor effect not accounting for the observed VP22 cell-to-cell spread in culture.
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Goldberg, Jacob M., Rebecca F. Wissner, Alyssa M. Klein, and E. James Petersson. "Correction: Thioamide quenching of intrinsic protein fluorescence." Chemical Communications 56, no. 25 (2020): 3699. http://dx.doi.org/10.1039/d0cc90120b.
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Meyer, Arne, Christian Betzel, and Marc Pusey. "Latest methods of fluorescence-based protein crystal identification." Acta Crystallographica Section F Structural Biology Communications 71, no. 2 (January 28, 2015): 121–31. http://dx.doi.org/10.1107/s2053230x15000114.
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Successful protein crystallization screening experiments are dependent upon the experimenter being able to identify positive outcomes. The introduction of fluorescence techniques has brought a powerful and versatile tool to the aid of the crystal grower. Trace fluorescent labeling, in which a fluorescent probe is covalently bound to a subpopulation (<0.5%) of the protein, enables the use of visible fluorescence. Alternatively, one can avoid covalent modification and use UV fluorescence, exploiting the intrinsic fluorescent amino acids present in most proteins. By the use of these techniques, crystals that had previously been obscured in the crystallization drop can readily be identified and distinguished from amorphous precipitate or salt crystals. Additionally, lead conditions that may not have been obvious as such under white-light illumination can be identified. In all cases review of the screening plate is considerably accelerated, as the eye can quickly note objects of increased intensity.
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Schlick, Kristian H., Candace K. Lange, Gregory D. Gillispie, and Mary J. Cloninger. "Characterization of Protein Aggregation via Intrinsic Fluorescence Lifetime." Journal of the American Chemical Society 131, no. 46 (November 25, 2009): 16608–9. http://dx.doi.org/10.1021/ja904073p.
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Duysak, Taner, Thanh Tuyen Tran, Aqeel Rana Afzal, and Che-Hun Jung. "Fluorescence Spectroscopic Analysis of ppGpp Binding to cAMP Receptor Protein and Histone-Like Nucleoid Structuring Protein." International Journal of Molecular Sciences 22, no. 15 (July 23, 2021): 7871. http://dx.doi.org/10.3390/ijms22157871.
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The cyclic AMP receptor protein (CRP) is one of the best-known transcription factors, regulating about 400 genes. The histone-like nucleoid structuring protein (H-NS) is one of the nucleoid-forming proteins and is responsible for DNA packaging and gene repression in prokaryotes. In this study, the binding of ppGpp to CRP and H-NS was determined by fluorescence spectroscopy. CRP from Escherichia coli exhibited intrinsic fluorescence at 341 nm when excited at 280 nm. The fluorescence intensity decreased in the presence of ppGpp. The dissociation constant of 35 ± 3 µM suggests that ppGpp binds to CRP with a similar affinity to cAMP. H-NS also shows intrinsic fluorescence at 329 nm. The fluorescence intensity was decreased by various ligands and the calculated dissociation constant for ppGpp was 80 ± 11 µM, which suggests that the binding site was occupied fully by ppGpp under starvation conditions. This study suggests the modulatory effects of ppGpp in gene expression regulated by CRP and H-NS. The method described here may be applicable to many other proteins.
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Ferreira, Sérgio T., and Tatiana Coelho-Sampaio. "Intrinsic fluorescence as a probe of structure-function relationships in Ca2+-transport ATPases." Bioscience Reports 16, no. 2 (April 1, 1996): 87–106. http://dx.doi.org/10.1007/bf01206199.
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Applications of intrinsic fluorescence measurements in the study of Ca2+-transport ATPases are reviewed. Since the initial reports showing that the fluorescence emission was sensitive to Ca2+ binding, a substantial amount of work has focused on the use of both steady-state and time-resolved fluorescence spectroscopy to investigate structure-function relationships in sarcoplasmic reticulum and plasma membrane Ca2+-ATPases. These studies have revealed ligand-induced conformational changes, as well as provided information on protein-protein, protein-solvent and/or protein-lipid interactions in different functional states of these proteins. The main results of these studies, as well as possible future prospects are discussed.
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Thaa, Bastian, Andreas Herrmann, and Michael Veit. "Intrinsic Cytoskeleton-Dependent Clustering of Influenza Virus M2 Protein with Hemagglutinin Assessed by FLIM-FRET." Journal of Virology 84, no. 23 (September 29, 2010): 12445–49. http://dx.doi.org/10.1128/jvi.01322-10.
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ABSTRACT The hemagglutinin (HA) of influenza virus organizes the virus bud zone, a domain of the plasma membrane enriched in raft lipids. Using fluorescence lifetime imaging microscopy-fluorescence resonance energy transfer (FLIM-FRET), a technique that detects close colocalization of fluorescent proteins in transfected cells, we show that the viral proton channel M2 clusters with HA but not with a marker for inner leaflet rafts. The FRET signal between M2 and HA depends on the raft-targeting signals in HA and on an intact actin cytoskeleton. We conclude that M2 contains an intrinsic signal that targets the protein to the viral bud zone, which is organized by raft-associated HA and by cortical actin.
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Stapelfeldt, Henrik, and Leif H. Skibsted. "Modification of β–lactoglobulin by aliphatic aldehydes in aqueous solution." Journal of Dairy Research 61, no. 2 (May 1994): 209–19. http://dx.doi.org/10.1017/s0022029900028223.
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SummaryEach of the secondary lipid oxidation products pentanal, hexanal and heptanal was found to react with β–lactoglobulin (β–lg) in a two-phase model System (aqueous phosphate buffer–1-octanol) yielding fluorescent condensation products (emission maximum, 410 nm; excitation maximum, 350 nm). Protein polymers were detected by size-exclusion HPLC, and the rate of reaction paralleled the formation of fluorescent products, with the reactivity being pentanal > hexanal > heptanal. Simultaneously, the reaction also changed the intrinsic fluorescence of β–lg, and in particular pentanal reduced the intensity of tryptophan fluorescence (emission maximum, 332 nm; excitation maximum, 288 nm) by 30%. These findings are discussed with reference to the effect of peroxidizing lipids on the physical properties of whey proteins and the use of protein fluorescence (induced by the reaction with aldehydes) as marker for the oxidative status of milk and whey protein products.
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Okerberg, Eric, and Jason B. Shear. "Attomole-Level Protein Fingerprinting Based on Intrinsic Peptide Fluorescence." Analytical Chemistry 73, no. 7 (April 2001): 1610–13. http://dx.doi.org/10.1021/ac0012703.
Full textDissertations / Theses on the topic "Intrinsic protein fluorescence"
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Paul, Uchenna Prince. "Fluorescence Detectors for Proteins and Toxic Heavy Metals." Diss., CLICK HERE for online access, 2004. http://contentdm.lib.byu.edu/ETD/image/etd416.pdf.
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Macmillan, Alexander Malcolm. "Bioencapsulation in silica sol-gel nano-pores and intrinsic protein fluorescence : ensemble and single molecule." Thesis, University of Strathclyde, 2009. http://oleg.lib.strath.ac.uk:80/R/?func=dbin-jump-full&object_id=25772.
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The ability to measure and understand protein fluorescence depends on the development of light sources which can excite the intrinsic aromatic amino acids, tryptophan, tyrosine, and phenylalanine. In recent years the time-resolved study of protein fluorescence has been limited to the excitation of tryptophan and tyrosine. The availability of the shorter wavelength, 265nm light source, allows for the excitation of phenylalanine which until recently has been limited. In this thesis the direct excitation of phenylalanine is demonstrated, using pulsed light emitting diodes, and the bi-exponential nature of its fluorescence decay is investigated, and the effect of pH on the fluorescence lifetimes. One of the major difficulties with the study of proteins is the lack of immobilisation techniques for the study of proteins at the single-molecule level, which provide little perturbation of the protein. To try to achieve this, the fabrication of novel molecular nanoenvironments, based on sol-gel techniques, which allow control and enhancement of protein fluorescence has been developed. In this thesis the application of sol-gel techniques is demonstrated for the environment sensitive trimeric form of allophycocyanin (APC) at both the ensemble and single-molecule level. The optimisation of the sol-gel technique as a generic approach to entrapment of proteins was developed using the environment sensitive probe 6-propionyl-2-(N,N-dimethylamino)naphthalene (PRODAN), which enabled monitoring of the hydrolysis and methanol removal stage of the process. For earlier diagnosis, ultra sensitive monitoring and breakthroughs in understanding the causes of many diseases, we urgently need to develop clinical single molecule sensing. Downstream, this might be accomplished by means of the fluorescence nanosecond/nanometre microscopy of single biomacromolecules.
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Niland, Hannah. "Detection and analysis of proteins in the solid phase using extrinsic and intrinsic fluorescence." Thesis, University of Edinburgh, 2017. http://hdl.handle.net/1842/28771.
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Over the past two decades a body of evidence concerning residual biological contamination on cleaned surgical instruments has accumulated. This is substantiated by the number of yearly surgery cancellations due to visible residue on instruments in surgical packs and incidences of iatrogenic Creutzfeldt-Jakob disease (iCJD). It is therefore imperative to develop a method of protein detection for use in clinical sterile services departments (SSDs) for monitoring of decontamination quality. This Thesis describes the development and use of an epifluorescence surface scanner (EFScan) technology in the assessment of proteinaceous residue on surgical instruments, by detecting protein pre-labelled with fluorescein isothiocyanate (FITC), and exploratory studies on the feasibility of label-free detection, using intrinsic protein fluorescence. Measurements using FITC labelling showed that residual protein on the order of micrograms can be found on new, single-use instruments (i.e. prior to use). This is comparable to the amount of residual protein found on retired, reusable instruments. To confirm the suitability of fluorescence techniques in the detection and quantification of proteinaceous residue, a blind, pilot study was carried out in conjunction with groups from Queen Mary University and the University of Southampton. Each University used a different labelling and detection method, and results showed good agreement between these methods. This showed that fluorescence is a suitable technique for the detection and quantification of proteinaceous contamination on surgical instruments. The next step in the project focussed on detection of contamination via intrinsic protein fluorescence from tryptophan residues, with a view to elimination of the labelling step. Intrinsic fluorescence of proteins in solution is widely characterised; however, fluorescence characteristics of solid or surface-bound protein have been little studied. Therefore, the characterisation of solid protein fluorescence and the emission characteristics of protein adsorbed onto stainless steel was undertaken. Analysis of the commonly used protein standard bovine serum albumin (BSA) showed that the two tryptophan residues it contains are highly susceptible to photo-oxidation in the solid state, resulting in conversion to the fluorescent photoproducts n-formylkynurenine (NFK) and kynurenine. Therefore, BSA is not suitable for use as a standard in the development of intrinsic fluorescence detection of surface-bound protein. The 72-tryptophan protein fibrinogen, as well as a series of other multi-tryptophan proteins, were assessed and it was found that photo-oxidation of the tryptophan residues did not occur on the irradiation timescale of 1 hour utilized. Therefore, it was concluded that lysozyme or gamma-globulins, a prominent group of serum proteins, would be more suitable candidates as a standard in subsequent research into the intrinsic detection and quantification of proteinaceous contamination. A third study explored the potential use of fluorescence in the early diagnosis of cataract. This involved the fluorescence characterisation of healthy porcine lenses and the use of UV irradiation of the lens to attempt to create cataract in vitro. There was found to be a large variation in fluorescence characteristics from lens to lens, suggesting that fluorophore concentrations can vary significantly. This implies that identification of a suitable standard for the early detection of cataract may be problematic. Attempts to create cataract resulted in the photo-oxidation pathway which had been observed in BSA, and although NFK and kynurenine play a role in cataractogenesis, the accumulation of these photoproducts is not analogous to a natural cataract. It was found that these products could be destroyed by irradiation of the lens at appropriate photo-bleaching wavelengths. However, this also destroyed intrinsic, protective fluorophores within the lens, suggesting that a light-based method of cataract treatment may not be achievable.
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Lepthien, Sandra. "In vivo tandem labeling of proteins combining chemical orthogonality with intrinsic blue fluorescence /." kostenfrei, 2008. http://mediatum2.ub.tum.de/node?id=673683.
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Hogue, Christopher Warren Victor. "Tryptophanyl-tRNA synthetase and its role in the incorporation of new intrinsic fluorescent probes into proteins." Thesis, University of Ottawa (Canada), 1994. http://hdl.handle.net/10393/10560.
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Fluorescence spectroscopy can reveal insights into the structure, function and dynamics of proteins using the intrinsic fluorescence of tryptophan (Trp) residues. Time-resolved fluorescence is uniquely sensitive to the local microenvironment of Trp residues. This methodology is widely used, yet extensions allowing the further study of protein interactions are desired. Tryptophan analogs are targeted as new intrinsic probes, since their structures may camouflage them sufficiently to trick the relevant protein synthesis mechanisms to allow their incorporation into proteins. The specificity of a single enzyme, tryptophanyl-tRNA synthetase (TrpRS; E.C. 6.1.1.2), is the key to success for biosynthetic Trp analog incorporation. TrpRS catalyzes the ATP activation of Trp, and the subsequent aminoacylation of tRNATrp, prior to ribosomal protein synthesis. Bacillus subtilis TrpRS has a single conserved and essential Trp-92 residue in each of its symmetrical alpha-2 subunits. The fluorescence of this Trp was very useful for investigating the TrpRS mechanism, together with the nonfluorescent isomorphous analog 4-fluorotryptophan as substrate. A tryptophanyl-5'-adenylate dependent quenching of Trp-92 fluorescence was observed, consistent with a local alpha-helix formation, placing a conserved Cys residue in quenching proximity to the Trp-92 fluorophore. This corresponds with the recent crystal structure of the homologous B. stearothermophilus TrpRS. Titrations while monitoring Trp-92 fluorescence revealed that the TrpRS dimer undergoes a concerted conformational change, virtually complete with the reaction of one subunit. This change seems necessary since this very small enzyme could only bind the relatively large tRNATrp substrate through interactions with both subunits. Potentially useful Trp analogs for fluorescence studies were examined to determine if they were TrpRS substrates. 5-hydroxytryptophan (5HW) and 7-azatryptophan (7AW) were studied both as TrpRS substrates, and as biosynthetically incorporated replacements of Trp-92. Methodology for efficient incorporation of these normally toxic Trp analogs is demonstrated. 7AW was shown to be very sensitive to solvent exposure, as its inclusion into a buried region of the protein causes large increases in its fluorescence yield. 7AW is recognized as having particular utility for the study of protein folding. Observations of the time-resolved fluorescence behaviour of Trp and 5HW in the same protein microenvironment are of consequence for fluorescence theory, as they indicate that the current assumptions regarding radiative properties may require modification. By combining insights from both the crystal structure and these intrinsic fluorescence studies with both Trp and the analogs, a clearer picture of the mechanism of TrpRS was obtained. Prior to this study, the role of the essential Trp-92 was not understood in terms of the mechanism of TrpRS. This study demonstrates Trp-92 is crucial for both TrpRS conformational stability and for its very dynamic mechanism which involves large, substrate-dependent conformational changes.
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McGuinness, Colin Douglas. "Glucose sensing based on the intrinsic time dependent fluorescence from proteins : application of pulsed ultraviolet light emitting diodes and sol-gel derived matrices." Thesis, University of Strathclyde, 2006. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.431823.
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Bourouah, Oussama. "Affinité et perturbation membranaire de la BSP1, une protéine du liquide séminal bovin: une étude avec des membranes lipidiques modèles." Thèse, 2020. http://hdl.handle.net/1866/23705.
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La BSP1, principale protéine du plasma séminal bovin, interagit avec les membranes des spermatozoïdes et joue un rôle crucial dans les événements qui conduisent à la fécondité des spermatozoïdes, lors du phénomène de la capacitation. Le but de cette recherche est d’investiguer la nature de ces interactions. Ce travail vise à démontrer l’influence des lipides qui composent les membranes sur l’action de la protéine BSP1. À l’aide de la fluorescence intrinsèque de la protéine, l’affinité de la protéine a été caractérisée pour quatre systèmes lipidiques. Les résultats montrent que la composition lipidique affecte significativement l'affinité de la protéine pour les membranes. Nous avons observé l'ordre suivant : 1-palmitoyl-2-oléoyl-sn-glycéro-3-phosphocholine (POPC) > POPC/1-palmitoyl-2-oléoyl-sn-glycéro-3-phosphoéthanolamine (POPE) ≈ POPC/1-palmitoyl-2-oléoyl-sn-glycéro-3-phospho-L-sérine (POPS) > POPC/cholestérol. La protéine interagit préférentiellement avec POPC. La présence de POPE, POPS, ou cholestérol dans la membrane diminue systématiquement l’affinité. Il est connu que la présence de POPE ou cholestérol augmente l’empilement des lipides dans les membranes. Cet effet de condensation des chaînes pourrait être défavorable à l’insertion de la partie hydrophobe de la protéine dans les membranes et réduire ainsi l'affinité. La diminution de l’affinité de la protéine induite par la présence de POPS, un lipide chargé négativement, pourrait être associée aux interactions électrostatiques répulsives car la protéine porte une charge globale négative.
La littérature mentionne que la BSP1 extrait sélectivement les phospholipides de type choline et le cholestérol lors de son association avec les membranes de spermatozoïdes. Un efflux lipidique est aussi observé avec des membranes modèles. Nous avons désiré caractériser la « solubilisation » des membranes par la BSP1, par diffusion dynamique de la lumière. Comme étape préliminaire, nous avons étudié comment le détergent Triton X-100 solubilise les membranes en utilisant cette technique. Les mesures démontrent que la composition lipidique des membranes (POPC, POPC/POPE, POPC/1-palmitoyl-2-oléoyl-sn-glycéro-3- [phospho-rac-(1-glycérol)] (POPG)) n’affecte pas le mécanisme général de solubilisation/reconstitution des membranes modèles. Il a été montré qu'il existe trois régions lors des processus de solubilisation pour les différents systèmes lipidiques : i) le détergent se distribue dans les membranes, ii) une coexistence de membranes saturées en détergents et de micelles mixtes de phospholipides/Triton X-100 et iii) exclusivement des micelles mixtes de phospholipides/Triton X-100. Nos résultats montrent que la forme conique de POPE augmente la résistance des membranes à la solubilisation. La présence de POPG, apportant une charge négative à l’interface des membranes, n’induit aucun changement aux processus de solubilisation/reconstitution des membranes par Triton X-100. La diffusion dynamique de la lumière a également permis d’observer si la protéine BSP1 induit des modifications morphologiques des membranes suite à son interaction avec les membranes de POPC. Nos observations n'ont montré aucune variation significative de la taille des particules lors du titrage des vésicules de POPC par la protéine, sur une gamme de rapport molaire de POPC/BSP1 variant de 20 à 0.6. Avec des compositions aussi différentes, on suppose une transition des vésicules saturées en protéine à des complexes de protéine avec un peu de lipides. Cependant, il semble impossible avec la diffusion dynamique de la lumière de différencier ces particules.BSP1, the main protein in bovine seminal plasma, interacts with sperm membranes and plays a crucial role in events that lead to sperm fertility, during the capacitation. The purpose of this research is to investigate the nature of these interactions. This work aims to demonstrate the influence of the lipids that compose membranes on the action of the BSP1 protein. Using the intrinsic fluorescence of the protein, the affinity of the protein was characterized for four lipid systems. The results show that the lipid composition significantly affects the affinity of the protein for membranes. We observed the following order: 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) > POPC/1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoethanolamine (POPE) ≈ POPC/1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoserine (POPS) > POPC/cholesterol. The protein interacts preferentially with POPC. The presence of POPE, POPS, or cholesterol in membranes decreases systematically the affinity. It is established that the presence of POPE or cholesterol increases the packing of lipids in membranes. This condensation effect could be detrimental to the insertion of the hydrophobic part of the protein into the membranes and reduces, as a consequence, the affinity. The decrease in protein affinity induced by the presence of POPS, a negatively charged lipid, could be associated with repulsive electrostatic interactions as the protein global charge is negative. The literature mentions that BSP1 selectively extracts choline phospholipids and cholesterol when combined with sperm membranes. A lipid efflux is also observed with model membranes. We characterized membrane "solubilisation" by BSP1, using dynamic light scattering. As a preliminary step, we studied how Triton X-100 detergent solubilizes membranes using this technique. The measurements showed that the lipid composition of the membranes does not affect the general solubilization/reconstitution mechanism of the model membranes (POPC, POPC/POPE, POPC/1-palmitoyl-2-oleoyl-sn-glycero-3-phospho-(1-rac-glycerol) (POPG)). It is known that three different regions exist during the solubilization process for the different lipid systems: i) the detergent is distributed in the membranes, ii) a coexistence of membranes saturated with detergents and mixed phospholipid/Triton X-100 micelles and iii) exclusively mixed phospholipid/Triton X-100 micelles. Our results show that the conical shape of POPE increases the resistance of the membranes to solubilization. The presence of POPG, bringing a negative charge at the membrane interface, does not induce any change in solubilization/reconstitution processes. Dynamic light scattering also made it possible to observe if the BSP1 protein induces morphological changes in the membranes following its interaction with POPC membranes. Our observations showed no significant variation in particle size during the titration of POPC vesicles by the protein, over a molar ratio range of POPC/BSP1 from 20 to 0.6. Considering such different compositions, a transition from vesicles saturated with protein to protein complexes with some lipids is assumed. However, it appeared impossible with dynamic light scattering to differentiate these particles.
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Lepthien, Sandra [Verfasser]. "In vivo tandem labeling of proteins : combining chemical orthogonality with intrinsic blue fluorescence / Sandra Lepthien." 2008. http://d-nb.info/992091578/34.
Full textBook chapters on the topic "Intrinsic protein fluorescence"
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Greulich, Karl Otto. "Intrinsic Fluorescence Techniques for Studies on Protein-Protein and Protein-RNA Interactions in RNP Particles." In RNP Particles, Splicing and Autoimmune Diseases, 48–71. Berlin, Heidelberg: Springer Berlin Heidelberg, 1998. http://dx.doi.org/10.1007/978-3-642-80356-7_3.
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Watt, Kate, and Iain J. McEwan. "Using Intrinsic Fluorescence Emission Spectroscopy to Study Steroid Receptor and Coactivator Protein Conformation Dynamics." In Methods in Molecular Biology, 205–18. Totowa, NJ: Humana Press, 2009. http://dx.doi.org/10.1007/978-1-60327-575-0_12.
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Neyroz, Paolo, and Stefano Ciurli. "Intrinsic Fluorescence of Intrinsically Disordered Proteins." In Methods in Molecular Biology, 435–40. Totowa, NJ: Humana Press, 2012. http://dx.doi.org/10.1007/978-1-61779-927-3_25.
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"Intrinsic Protein Fluorescence." In Introduction to Fluorescence, 269–94. CRC Press, 2014. http://dx.doi.org/10.1201/b16502-15.
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Chan, Fiona T. S., Dorothea Pinotsi, S. Gabriele, Kaminski Schierle, and Clemens F. Kaminski. "Structure-Specific Intrinsic Fluorescence of Protein Amyloids Used to Study their Kinetics of Aggregation." In Bio-nanoimaging, 147–55. Elsevier, 2014. http://dx.doi.org/10.1016/b978-0-12-394431-3.00013-4.
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Eftink, Maurice R., and Haripada Maity. "Use of optical spectroscopic methods to study the thermodynamic stability of proteins." In Spectrophotometry and Spectrofluorimetry. Oxford University Press, 2000. http://dx.doi.org/10.1093/oso/9780199638130.003.0016.
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The biophysical characterization of globular proteins will almost always include some type of study of the unfolding of protein to obtain thermodynamic parameters. The basic idea is that a transition between a native and unfolded state, induced by temperature, pH, or denaturant concentration, can serve as a standard reaction for obtaining a thermodynamic measure of the stability of the native state. For example, the free energy change for the unfolding reaction can be used to compare the stability of a set of mutant forms of a protein (1-4). This type of analysis is based both on assumptions of the thermodynamic model for the unfolding process and on assumptions in the way the data are analysed; some of these assumptions and their limitations will be discussed below. There are a variety of methods that can be used to monitor an unfolding process. A common method is differential scanning calorimetry, DSC, which measures the variation in the specific heat of a protein-containing solution as a protein is thermally unfolded (5-7). DSC is a popular method for this purpose, but optical methods can also provide suitable information for tracking the unfolding of a protein The spectroscopic signals for the native and unfolded states of a protein can give some insight regarding the structure of the states, and often can provide advantages of economy, ease of measurement and amenability to a wide range of sample concentration. The optical spectroscopic methods that have been used most often for this purpose are absorption spectroscopy, circular dichroism and fluorescence, which will be discussed in this chapter. A key to each of these methods and their use in protein unfolding studies is that the signal is a mole fraction weighted average of the signals of each thermodynamic state. That is, the observed signal, S, can be expressed as . . . S = ∑XiSi . . . . . . 1 . . . where Xi is the mole fraction of species i and si is the intrinsic signal of species i. In order for a particular spectroscopic signal to be useful for tracking a N ↔ U transition of a protein, the signal must be sufficiently different for the N and U states.
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Karlish, S. J. D. "[24] Use of formycin nucleotides, intrinsic protein fluorescence, and fluorescein isothiocyanate-labeled enzymes for measurement of conformational states of Na+,K+-ATPase." In Methods in Enzymology, 271–77. Elsevier, 1988. http://dx.doi.org/10.1016/0076-6879(88)56027-5.
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Graves, Steven W., and John P. Nolan. "Molecular Assemblies, Probes, and Proteomics in Flow Cytometry." In Flow Cytometry for Biotechnology. Oxford University Press, 2005. http://dx.doi.org/10.1093/oso/9780195183146.003.0013.
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The many proteins and nucleic acids encoded in the genome predominantly perform their functions as macromolecular assemblies. In fact, modern biomedical research often targets the interactions of individual molecules of these assemblies, usually by disrupting or enhancing specific contacts, to provide treatment for many different diseases. Therefore, efficient pharmaceutical design requires knowledge of how macromolecular assemblies are built and function. To achieve this goal, sensitive and quantitative tools are essential. This chapter will discuss the use of flow cytometry as a general platform for sensitive measurement and quantification of molecular assemblies. First, this chapter will introduce general methods for analysis of molecular interactions along with a comparison of flow cytometry with these methods. Second, an overview of current flow cytometry instrumentation, assay technologies, and applications in molecular assembly analysis will be given. Third, the implementation of the above approaches in molecular assembly will be discussed. Finally, potential future directions of flow cytometry in molecular assembly analysis will be explored. At present, the analysis of macromolecular assemblies is performed by a wide variety of techniques that are chosen for the target molecules under study (proteins, DNA, lipids, etc.), the type of measurement required (kinetic or equilibrium), and whether the assembly of interest needs to be studied in vivo or in vitro. This continuum of techniques can be divided into the heterogeneous assays, which require a separation step to resolve products from reactants, and homogeneous assays, which can measure interactions without a separation step. Heterogeneous assays, in general, use radioisotopes, which are not perturbing; offer excellent sensitivity; and provide accurate quantification. The products are quantified after a separation step such as gel filtration, gel electrophoresis, or centrifugation. Rapid quench methods can provide subsecond kinetic resolution; however, the added separation steps are tedious and make collection of kinetic time courses difficult, as each time point must be separated and measured individually. Furthermore, in the time it takes the separation to occur, the interaction of interest can dissociate, which is a problem specific to low-affinity assemblies. Nonetheless, by using rapid chemical quench techniques, reaction times as short as a few milliseconds can be observed. Homogenous assays can be separated into solution- or surface-based assays. Solutionbased assays measure an optical signal generated by the assembly to quantify an interaction. High component concentrations (micromolar) allow changes in intrinsic molecular properties, such as protein fluorescence or circular dichroism, to be used to study molecular assemblies. For greater sensitivity (nanomole component concentrations), resonance energy transfer or polarization assays using exogenous fluorescent labels can be used. In combination with stopped-flow spectroscopy methodologies, solution-based assays allow reactions to monitored in a continuous fashion with submillisecond dead times.
Conference papers on the topic "Intrinsic protein fluorescence"
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Mosca, A., S. Viganò D'Angelo, and A. D'Angelo. "ACTIVATION OF PROTEIN C INDUCES CHANGES IN ITS INTRINSIC FLUORESCENCE." In XIth International Congress on Thrombosis and Haemostasis. Schattauer GmbH, 1987. http://dx.doi.org/10.1055/s-0038-1644302.
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Upon activation with either thrombin (T) or thrombin-thrombo modulin complex (T-TM), the zymogen protein C (PC) is transformed into a serine-protease, activated protein C(APC), by release of a small activation peptide. The rate of PC activation changes dramatically with T or with T-TM as a function of the Ca++ concentration in the activation medium, suggesting a configurational change of the zymogen in the presence of Ca++ . It has been shown that Ca++ binding to one single high-affinity binding site of gla-domainless PC is accompanied by a significant decrease of the intrinsic fluo rescence emission intensity of the protein and that the high-affinity binding site is retained following activation of gla-domain-less PC (J. Biol. Chem: 258; 5554, 1983). In the present work we have investigated the fluorescence properties of PC in order to answer the following questions: 1) is there a difference in the fluorescence properties of PC as compared to APC? 2) is there a difference between the conformational changes of PC activated with T or T-TM? From our experimental data we conclude that : a) the fluorescence emission intensity of fully activated PC is about 54% of the PC zymogen fluorescence intensity (λexc 280 nm, λ em 345 nm, 0.6 μuM PC or APC in 20 mMTris-HCl, 0.1 M NaCl, pH 7.8 at 25°C) ; b) during activation of PC (2 μM) with T-TM (150 nM) in the presence of 2 mM Ca++ , there is a good correlation (r=0.959) between fluorescence quenching and degree of PC activation, as measured by the rate of cleavage of the chromogenic substrate S-2238; c) the maximal fluorescence quench of PC activated with T or T-TM are virtually identical. Preliminary data suggest that Ca++ affects differently the fluorescence emission properties of PC and APC. These results suggest that evaluation of the fluorescence properties of PC might represent a valuable tool for the characterization of abnormal PC molecules.
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Einstein, Gnanatheepam, Kanniyappan Udayakumar, Prakasarao Aruna, and Singaravelu Ganesan. "Intrinsic fluorescence of protein in turbid media using empirical relation based on Monte Carlo lookup table." In SPIE BiOS, edited by Valery V. Tuchin, Kirill V. Larin, Martin J. Leahy, and Ruikang K. Wang. SPIE, 2017. http://dx.doi.org/10.1117/12.2255883.
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Akbay, Nuriye, Joseph R. Lakowicz, and Krishanu Ray. "Distance-dependent intrinsic fluorescence of proteins on aluminum nanostructures." In SPIE BiOS, edited by Tuan Vo-Dinh and Joseph R. Lakowicz. SPIE, 2012. http://dx.doi.org/10.1117/12.928379.
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Ray, Krishanu, Henryk Szmacinski, Mustafa H. Chowdhury, and Joseph R. Lakowicz. "Metal-enhanced intrinsic fluorescence of proteins and label-free bioassays." In BiOS, edited by Tuan Vo-Dinh and Joseph R. Lakowicz. SPIE, 2010. http://dx.doi.org/10.1117/12.840412.
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Yapoudjian, S., M. Ivanova, Olivier P. Uteza, Vladimir I. Marine, and Marc L. Sentis. "Surface intrinsic fluorescence spectroscopy of proteins using a UV linearly polarized pulsed laser beam." In International Conference on Atomic and Molecular Pulsed Lasers III, edited by Victor F. Tarasenko, Georgy V. Mayer, and Gueorgii G. Petrash. SPIE, 2000. http://dx.doi.org/10.1117/12.383460.
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Nielsen, Mogens Brøndsted, Michael Axman Petersen, Lars H. Andersen, V. K. Vaidyan, and V. S. Jayakumar. "Synthesis and intrinsic optical properties of retinal Schiff base and Green Fluorescent Protein chromophores." In PERSPECTIVES IN VIBRATIONAL SPECTROSCOPY: Proceedings of the 2nd International Conference on Perspectives in Vibrational Spectroscopy (ICOPVS 2008). AIP, 2008. http://dx.doi.org/10.1063/1.3046220.
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Martinez, Gloria M., Lauren S. Gollahon, Keri Shafer, Sowmini K. Oomman, Christian Busch, and Raul Martinez-Zaguilan. "Fluorescent pH probes, fluorescent proteins, and intrinsic cellular fluorochromes are tools to study cytosolic pH (pHcyt) in mammalian cells." In BiOS 2001 The International Symposium on Biomedical Optics, edited by Gregory H. Bearman, Darryl J. Bornhop, and Richard M. Levenson. SPIE, 2001. http://dx.doi.org/10.1117/12.432492.
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Daino, Michael M., and Satish G. Kandlikar. "Evaluation of Imaging Techniques Applied to Water Management Research in PEMFCs." In ASME 2009 7th International Conference on Nanochannels, Microchannels, and Minichannels. ASMEDC, 2009. http://dx.doi.org/10.1115/icnmm2009-82031.
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Water management in proton exchange membrane fuel cells (PEMFCs) is critical in efficient operation of fuel cells during normal operation as well as purge and start-up conditions. Insufficient membrane hydration impedes the flow of protons and an overabundance of water obstructs the flow of reactants in the gas diffusion layer (GDL) and in gas distribution channels. These two extremes of water content in PEMFCs significantly reduce performance and efficiency, causing material degradation and potential failure. Visualization and quantitative measurement of water content in PEMFCs lead to greater comprehension of water distribution and transport processes. A wide variety of imaging techniques have been employed in literature to reveal water distribution and transport processes on both macroscale and microscale. The presented techniques utilize visible, infrared, X-rays, fluorescence microscopy, nuclear magnetic resonance (NMR), and neutron radiography to visualize water, measure temperature distributions, and quantify water content. Each imaging technique has intrinsic advantages, disadvantages, and limitations for water detection and will be discussed. A critical evaluation of these techniques and their suitability for visualization of specific components of PEMFC are also discussed.
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Ren, Z. F. "Nano Materials and Physics." In ASME 4th Integrated Nanosystems Conference. ASMEDC, 2005. http://dx.doi.org/10.1115/nano2005-87045.
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Aligning carbon nanotubes in any way desired is very important for many fundamental and applied research projects. In this talk, I will first discuss how to grow them with controlled diameter, length, spacing, and periodicity using catalyst prepared by magnetron sputtering, electron beam (e-beam) lithography, electrochemical deposition, and nanosphere self-assembly. Then I will present our results of field emission property of both the aligned carbon nanotubes grown on flat substrates and random carbon nanotubes grown on carbon cloth. For the aligned carbon nanotubes arrays, I will present the preliminary results of using them as photonic band gap crystals and nanoantennae. As an alternative material of carbon nanotubes, ZnO nanowires have been grown in both aligned fashion on flat substrates and random fashion on carbon cloth. Using these ZnO nanowires, good field emission properties were observed. Furthermore, I will present our recent studies on the electrical breakdown and transport properties of a single suspended nanotube grown on carbon cloth by a scanning electron microscope probe incorporated into a high resolution transmission electron microscope. As part of the potential applications, I will also discuss our recent success on molecules delivery into cells using carbon nanotubes. Finally I will talk about our most recent endeavor on achieving thermoelectric figure-of-merit (ZT) higher than 2 using our unique nanocomposite approach. Plasma-enhanced chemical vapor deposition (PECVD) was discovered by my group in 1998 to be able to grow aligned carbon nanotubes [1]. Catalyst film was first deposited by magnetron sputtering. According to the thickness of the catalytic film, aligned carbon nanotubes were grown with different diameters and spacing, and different length depending the growth time. However, the two major drawbacks are 1) that the location of where the nantoube grows can not be controlled, 2) that the spacing between the nanotubes can not be varied too much. Therefore, we immediately explored to grow aligned carbon nanotubes with the location and spacing controls using e-beam lithography [2]. Unfortunately the cost is so high that the e-beam is not suited for large scale commercialization that requires only an average site density control not the exactly location, for example, electron source. It is the cost issue that made us to develop electrochemical deposition to make catalyst dots that can be separated more than 10 micormeters between dots [3]. With such arrays, we tested many samples for field emission properties and found the optimal site density [4]. However, for applications that require the location controls, for example, photonic band gap crystals, electrochemical deposition can not be satisfactory. It is this kind of application that led us to develop the nanosphere self-assembly technique in large scale [5]. For field emission, we found that ZnO nanowires are good field emitters comparable to carbon nanotubes if they are grown with the right diameter and spacing. Here I will discuss the field emission properties of ZnO nanowires as an alternative material to carbon nanotubes [6]. Us a special kind of carbon nanotubes made by PECVD, we discovered a highly efficient molecular delivery technique, named nanotube spearing, based on the penetration of Ni-particle embedded nanotubes into cell membranes by magnetic field driving. DNA plasmids encoding the enhanced green fluorescent protein (EGFP) sequence were immobilized onto the nanotubes, and subsequently speared into targeted cells. We have achieved the unprecedented high transduction efficiency in Bal17 B-lymphoma, ex vivo B cells, and primary neurons with high viability. This technique may provide a powerful tool for high efficient gene transfer in a variety of cells, especially, the hard-to-transfect cells [7]. Conventional transport studies of multiwall carbon nanotubes (MWNTs) with only the outmost wall contacted to the electrodes via side-contact shows that a MWNT is a ballistic conductor with only the outmost wall carrying current. Here we show, by using end-contact in which every wall is contacted to the electrodes, that every wall is conducting, as evidenced by a significant amount of current drop when an innermost wall is broken at high-bias. Remarkably, the breakdown of each wall was initiated in the middle of the nanotube, not at the contacts, indicating diffusive electron transport. Using end-contact, we were able to probe the conductivity wall-by-wall and found that each wall is indeed either metallic, or semiconducting, or pseudogap-like. These findings not only reveal the intrinsic physical properties of MWNTs but also provide important guidance to MWNT-based electronic devices [8]. At the end of the talk, if time permits, I will talk about our ongoing effort on improving the figure-of-merit (ZT) of thermoelectric materials using a nanocomposite strategy to mimic the structure of the superlattice of PbTe/PbSe and Bi2Te3/Sb2Te3 hoping to reduce the thermal conductivity by a factor of 2–4 while maintaining the electrical conductivity. To make a close to 100% dense nanocomposite, we started with nanoparticles synthesis, then consolidation using both the traditional hot press and the direct current fast-heat, named plasma pressure compact, to preserve the nano size of the component particles. So far, we have seen thermal conductivity decrease by a factor of 2 in the systems of Si/Ge, PbeTe/PbSe, Bi2Te3/Sb2Te3, indicating the potential of improving ZT by a factor of 2.
Reports on the topic "Intrinsic protein fluorescence"
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Epel, Bernard, and Roger Beachy. Mechanisms of intra- and intercellular targeting and movement of tobacco mosaic virus. United States Department of Agriculture, November 2005. http://dx.doi.org/10.32747/2005.7695874.bard.
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To cause disease, plant viruses must replicate and spread locally and systemically within the host. Cell-to-cell virus spread is mediated by virus-encoded movement proteins (MPs), which modify the structure and function of plasmodesmata (Pd), trans-wall co-axial membranous tunnels that interconnect the cytoplasm of neighboring cells. Tobacco mosaic virus (TMV) employ a single MP for cell- cell spread and for which CP is not required. The PIs, Beachy (USA) and Epel (Israel) and co-workers, developed new tools and approaches for study of the mechanism of spread of TMV that lead to a partial identification and molecular characterization of the cellular machinery involved in the trafficking process. Original research objectives: Based on our data and those of others, we proposed a working model of plant viral spread. Our model stated that MPᵀᴹⱽ, an integral ER membrane protein with its C-terminus exposed to the cytoplasm (Reichel and Beachy, 1998), alters the Pd SEL, causes the Pd cytoplasmic annulus to dilate (Wolf et al., 1989), allowing ER to glide through Pd and that this gliding is cytoskeleton mediated. The model claimed that in absence of MP, the ER in Pd (the desmotubule) is stationary, i.e. does not move through the Pd. Based on this model we designed a series of experiments to test the following questions: -Does MP potentiate ER movement through the Pd? - In the presence of MP, is there communication between adjacent cells via ER lumen? -Does MP potentiate the movement of cytoskeletal elements cell to cell? -Is MP required for cell-to-cell movement of ER membranes between cells in sink tissue? -Is the binding in situ of MP to RNA specific to vRNA sequences or is it nonspecific as measured in vitro? And if specific: -What sequences of RNA are involved in binding to MP? And finally, what host proteins are associated with MP during intracellular targeting to various subcellular targets and what if any post-translational modifications occur to MP, other than phosphorylation (Kawakami et al., 1999)? Major conclusions, solutions and achievements. A new quantitative tool was developed to measure the "coefficient of conductivity" of Pd to cytoplasmic soluble proteins. Employing this tool, we measured changes in Pd conductivity in epidermal cells of sink and source leaves of wild-type and transgenic Nicotiana benthamiana (N. benthamiana) plants expressing MPᵀᴹⱽ incubated both in dark and light and at 16 and 25 ᵒC (Liarzi and Epel, 2005 (appendix 1). To test our model we measured the effect of the presence of MP on cell-to-cell spread of a cytoplasmic fluorescent probe, of two ER intrinsic membrane protein-probes and two ER lumen protein-probes fused to GFP. The effect of a mutant virus that is incapable of cell-to-cell spread on the spread of these probes was also determined. Our data shows that MP reduces SEL for cytoplasmic molecules, dilates the desmotubule allowing cell-cell diffusion of proteins via the desmotubule lumen and reduces the rate of spread of the ER membrane probes. Replicase was shown to enhance cell-cell spread. The data are not in support of the proposed model and have led us to propose a new model for virus cell-cell spread: this model proposes that MP, an integral ER membrane protein, forms a MP:vRNAER complex and that this ER-membrane complex diffuses in the lipid milieu of the ER into the desmotubule (the ER within the Pd), and spreads cell to cell by simple diffusion in the ER/desmotubule membrane; the driving force for spread is the chemical potential gradient between an infected cell and contingent non-infected neighbors. Our data also suggests that the virus replicase has a function in altering the Pd conductivity. Transgenic plant lines that express the MP gene of the Cg tobamovirus fused to YFP under the control the ecdysone receptor and methoxyfenocide ligand were generated by the Beachy group and the expression pattern and the timing and targeting patterns were determined. A vector expressing this MPs was also developed for use by the Epel lab . The transgenic lines are being used to identify and isolate host genes that are required for cell-to-cell movement of TMV/tobamoviruses. This line is now being grown and to be employed in proteomic studies which will commence November 2005. T-DNA insertion mutagenesis is being developed to identify and isolate host genes required for cell-to-cell movement of TMV.