RESUMEN
Experimental methods to determine transition temperatures for individual base pair melting events in DNA duplexes are lacking despite intense interest in these thermodynamic parameters. Here, we determine the dimensions of the thymine (T) C2âO stretching vibration when it is within the DNA duplex via isotopic substitutions at other atomic positions in the structure. First, we determined that this stretching state was localized enough to specific atoms in the molecule to make submolecular scale measurements of local structure and stability in high molecular weight complexes. Next, we develop a new isotope-edited variable temperature infrared method to measure melting transitions at various locations in a DNA structure. As an initial test of this "sub-molecular scale thermometer", we applied our T13C2 difference infrared signal to measure location-dependent melting temperatures (TmL) in a DNA duplex via variable temperature attenuated total reflectance Fourier transform infrared (VT-ATR-FTIR) spectroscopy. We report that the TmL of a single Watson-Crick A-T base pair near the end of an A-T rich sequence (poly T) is â¼34.9 ± 0.7°C. This is slightly lower than the TmL of a single base pair near the middle position of the poly T sequence (TmL â¼35.6±0.2°C). In addition, we also report that the TmL of a single Watson-Crick A-T base pair near the end of a 50% G-C sequence (12-mer) is â¼52.5 ± 0.3°C, which is slightly lower than the global melting Tm of the 12-mer sequence (TmL â¼54.0±0.9°C). Our results provide direct physical evidence for end fraying in DNA sequences with our novel spectroscopic methods.
Asunto(s)
Emparejamiento Base , ADN , Timina , Temperatura de Transición , ADN/química , Timina/química , Espectroscopía Infrarroja por Transformada de Fourier , Espectrofotometría Infrarroja/métodos , Conformación de Ácido Nucleico , TemperaturaRESUMEN
The human copper-binding protein metallothionein-3 (MT-3) can reduce Cu(II) to Cu(I) and form a polynuclear Cu(I)4-Cys5-6 cluster concomitant with intramolecular disulfide bonds formation, but the cluster is unusually inert toward O2 and redox-cycling. We utilized a combined array of rapid-mixing spectroscopic techniques to identify and characterize the transient radical intermediates formed in the reaction between Zn7MT-3 and Cu(II) to form Cu(I)4Zn(II)4MT-3. Stopped-flow electronic absorption spectroscopy reveals the rapid formation of transient species with absorption centered at 430-450 nm and consistent with the generation of disulfide radical anions (DRAs) upon reduction of Cu(II) by MT-3 cysteine thiolates. These DRAs are oxygen-stable and unusually long-lived, with lifetimes in the seconds regime. Subsequent DRAs reduction by Cu(II) leads to the formation of a redox-inert Cu(I)4-Cys5 cluster with short Cu-Cu distances (<2.8 Å), as revealed by low-temperature (77 K) luminescence spectroscopy. Rapid freeze-quench Raman and electron paramagnetic resonance (EPR) spectroscopy characterization of the intermediates confirmed the DRA nature of the sulfur-centered radicals and their subsequent oxidation to disulfide bonds upon Cu(II) reduction, generating the final Cu(I)4-thiolate cluster. EPR simulation analysis of the radical g- and A-values indicate that the DRAs are directly coupled to Cu(I), potentially explaining the observed DRA stability in the presence of O2. We thus provide evidence that the MT-3 Cu(I)4-Cys5 cluster assembly process involves the controlled formation of novel long-lived, copper-coupled, and oxygen-stable disulfide radical anion transient intermediates.
Asunto(s)
Cobre/química , Disulfuros/química , Radicales Libres/química , Metalotioneína 3/química , Oxígeno/química , Espectroscopía de Resonancia por Spin del Electrón , Glutatión/química , Humanos , Metalotioneína 3/genética , Metalotioneína 3/metabolismo , Oxidación-Reducción , Proteínas Recombinantes/biosíntesis , Proteínas Recombinantes/química , Proteínas Recombinantes/aislamiento & purificación , Espectrometría de Fluorescencia , Zinc/químicaRESUMEN
Three-dimensional chitinous scaffolds often used in regenerative medicine, tissue engineering, biomimetics and technology are mostly isolated from marine organisms, such as marine sponges (Porifera). In this work, we report the results of the electrochemical isolation of the ready to use chitinous matrices from three species of verongiid demosponges (Aplysina archeri, Ianthella basta and Suberea clavata) as a perfect example of possible morphological and chemical dimorphism in the case of the marine chitin sources. The electrolysis of concentrated Na2SO4 aqueous solution showed its superiority over the chemical chitin isolation method in terms of the treatment time reduction: only 5.5 h for A. archeri, 16.5 h for I. basta and 20 h for the S. clavata sample. Further investigation of the isolated scaffolds by digital microscopy and SEM showed that the electrolysis-supported isolation process obtains chitinous scaffolds with well-preserved spatial structure and it can be competitive to other alternative chitin isolation techniques that use external accelerating factors such as microwave irradiation or atmospheric plasma. Moreover, the infrared spectroscopy (ATR-FTIR) proved that with the applied electrochemical conditions, the transformation into chitosan does not take place.
Asunto(s)
Quitina , Poríferos , Animales , Quitina/química , Espectroscopía Infrarroja por Transformada de Fourier , Poríferos/química , ElectrólisisRESUMEN
Marine sponges were among the first multicellular organisms on our planet and have survived to this day thanks to their unique mechanisms of chemical defense and the specific design of their skeletons, which have been optimized over millions of years of evolution to effectively inhabit the aquatic environment. In this work, we carried out studies to elucidate the nature and nanostructural organization of three-dimensional skeletal microfibers of the giant marine demosponge Ianthella basta, the body of which is a micro-reticular, durable structure that determines the ideal filtration function of this organism. For the first time, using the battery of analytical tools including three-dimensional micro-X-ray Fluorescence (3D-µXRF), X-ray diffraction (XRD), infra-red (FTIR), Raman and Near Edge X-ray Fine Structure (NEXAFS) spectroscopy, we have shown that biomineral calcite is responsible for nano-tuning the skeletal fibers of this sponge species. This is the first report on the presence of a calcitic mineral phase in representatives of verongiid sponges which belong to the class Demospongiae. Our experimental data suggest a possible role for structural amino polysaccharide chitin as a template for calcification. Our study suggests further experiments to elucidate both the origin of calcium carbonate inside the skeleton of this sponge and the mechanisms of biomineralization in the surface layers of chitin microfibers saturated with bromotyrosines, which have effective antimicrobial properties and are responsible for the chemical defense of this organism. The discovery of the calcified phase in the chitinous template of I. basta skeleton is expected to broaden the knowledge in biomineralization science where the calcium carbonate is regarded as a valuable material for applications in biomedicine, environmental science, and even in civil engineering.
Asunto(s)
Organismos Acuáticos/química , Carbonato de Calcio/química , Poríferos/química , Esqueleto/química , Animales , Biomineralización , Quitina/química , Espectroscopía Infrarroja por Transformada de Fourier , Andamios del Tejido/química , Difracción de Rayos XRESUMEN
Structure-based tissue engineering requires large-scale 3D cell/tissue manufacture technologies, to produce biologically active scaffolds. Special attention is currently paid to naturally pre-designed scaffolds found in skeletons of marine sponges, which represent a renewable resource of biomaterials. Here, an innovative approach to the production of mineralized scaffolds of natural origin is proposed. For the first time, a method to obtain calcium carbonate deposition ex vivo, using living mollusks hemolymph and a marine-sponge-derived template, is specifically described. For this purpose, the marine sponge Aplysin aarcheri and the terrestrial snail Cornu aspersum were selected as appropriate 3D chitinous scaffold and as hemolymph donor, respectively. The formation of calcium-based phase on the surface of chitinous matrix after its immersion into hemolymph was confirmed by Alizarin Red staining. A direct role of mollusks hemocytes is proposed in the creation of fine-tuned microenvironment necessary for calcification ex vivo. The X-ray diffraction pattern of the sample showed a high CaCO3 amorphous content. Raman spectroscopy evidenced also a crystalline component, with spectra corresponding to biogenic calcite. This study resulted in the development of a new biomimetic product based on ex vivo synthetized ACC and calcite tightly bound to the surface of 3D sponge chitin structure.
Asunto(s)
Quitina/análogos & derivados , Quitina/química , Hemolinfa/metabolismo , Poríferos/metabolismo , Caracoles/metabolismo , Andamios del Tejido , Animales , Biomineralización , Carbonato de Calcio/química , Difracción de Rayos XRESUMEN
A( syn)-T and G( syn)-C+ Hoogsteen base pairs in protein-bound DNA duplexes can be difficult to resolve by X-ray crystallography due to ambiguous electron density and by nuclear magnetic resonance (NMR) spectroscopy due to poor chemical shift dispersion and size limitations with solution-state NMR spectroscopy. Here we describe an NMR strategy for characterizing Hoogsteen base pairs in protein-DNA complexes, which relies on site-specifically incorporating 13C- and 15N-labeled nucleotides into DNA duplexes for unambiguous resonance assignment and to improve spectral resolution. The approach was used to resolve the conformation of an A-T base pair in a crystal structure of an â¼43 kDa complex between a 34 bp duplex DNA and the integration host factor (IHF) protein. In the crystal structure (Protein Data Bank entry 1IHF ), this base pair adopts an unusual Hoogsteen conformation with a distorted sugar backbone that is accommodated by a nearby nick used to aid in crystallization. The NMR chemical shifts and interproton nuclear Overhauser effects indicate that this base pair predominantly adopts a Watson-Crick conformation in the intact DNA-IHF complex under solution conditions. Consistent with these NMR findings, substitution of 7-deazaadenine at this base pair resulted in only a small (â¼2-fold) decrease in the IHF-DNA binding affinity. The NMR strategy provides a new approach for resolving crystallographic ambiguity and more generally for studying the structure and dynamics of protein-DNA complexes in solution.
Asunto(s)
Emparejamiento Base , Proteínas de Unión al ADN/química , ADN/química , Sustancias Macromoleculares/química , Espectroscopía de Resonancia Magnética/métodos , Conformación de Ácido Nucleico , Secuencia de Bases , Isótopos de Carbono/metabolismo , Cristalografía por Rayos X , ADN/genética , ADN/metabolismo , Proteínas de Unión al ADN/metabolismo , Sustancias Macromoleculares/metabolismo , Modelos Moleculares , Estructura Molecular , Isótopos de Nitrógeno/metabolismo , Nucleótidos/química , Nucleótidos/genética , Nucleótidos/metabolismo , Dominios ProteicosRESUMEN
Hoogsteen DNA base pairs (bps) are an alternative base pairing to canonical Watson-Crick bps and are thought to play important biochemical roles. Hoogsteen bps have been reported in a handful of X-ray structures of protein-DNA complexes. However, there are several examples of Hoogsteen bps in crystal structures that form Watson-Crick bps when examined under solution conditions. Furthermore, Hoogsteen bps can sometimes be difficult to resolve in DNA:protein complexes by X-ray crystallography due to ambiguous electron density and by solution-state NMR spectroscopy due to size limitations. Here, using infrared spectroscopy, we report the first direct solution-state observation of a Hoogsteen (G-C+ ) bp in a DNA:protein complex under solution conditions with specific application to DNA-bound TATA-box binding protein. These results support a previous assignment of a G-C+ Hoogsteen bp in the complex, and indicate that Hoogsteen bps do indeed exist under solution conditions in DNA:protein complexes.
Asunto(s)
Citosina/química , ADN/metabolismo , Guanina/química , Proteína de Unión a TATA-Box/metabolismo , Emparejamiento Base , Cristalografía por Rayos X , ADN/química , Conformación de Ácido Nucleico , Espectrofotometría Infrarroja , Proteína de Unión a TATA-Box/químicaRESUMEN
Innovative materials were made via the combination of chitin and lignin, and the immobilization of lipase from Aspergillus niger. Analysis by techniques including FTIR, XPS and 13C CP MAS NMR confirmed the effective immobilization of the enzyme on the surface of the composite support. The electrokinetic properties of the resulting systems were also determined. Results obtained from elemental analysis and by the Bradford method enabled the determination of optimum parameters for the immobilization process. Based on the hydrolysis reaction of para-nitrophenyl palmitate, a determination was made of the catalytic activity, thermal and pH stability, and reusability. The systems with immobilized enzymes were found to have a hydrolytic activity of 5.72 mU, and increased thermal and pH stability compared with the native lipase. The products were also shown to retain approximately 80% of their initial catalytic activity, even after 20 reaction cycles. The immobilization process, using a cheap, non-toxic matrix of natural origin, leads to systems with potential applications in wastewater remediation processes and in biosensors.
Asunto(s)
Aspergillus niger/enzimología , Quitina/química , Enzimas Inmovilizadas/metabolismo , Proteínas Fúngicas/metabolismo , Lignina/química , Lipasa/metabolismo , Técnicas Biosensibles , Fenómenos Químicos , Restauración y Remediación Ambiental , Estabilidad de Enzimas , Enzimas Inmovilizadas/química , Proteínas Fúngicas/química , Concentración de Iones de Hidrógeno , Hidrólisis , Cinética , Lipasa/química , Ensayo de Materiales , Resonancia Magnética Nuclear Biomolecular , Palmitatos/metabolismo , Espectroscopía de Fotoelectrones , Espectroscopía Infrarroja por Transformada de Fourier , Temperatura , Calidad del AguaRESUMEN
S-adenosyl-L-methionine (SAM) is an abundant biomolecule used by methyltransferases to regulate a wide range of essential cellular processes such as gene expression, cell signaling, protein functions, and metabolism. Despite considerable effort, there remain many specificity challenges associated with designing small molecule inhibitors for methyltransferases, most of which exhibit off-target effects. Interestingly, NMR evidence suggests that SAM undergoes conformeric exchange between several states when free in solution. Infrared spectroscopy can detect different conformers of molecules if present in appreciable populations. When SAM is noncovalently bound within enzyme active sites, the nature and the number of different conformations of the molecule are likely to be altered from when it is free in solution. If there are unique structures or different numbers of conformers between different methyltransferase active sites, solution-state information may provide promising structural leads to increase inhibitor specificity for a particular methyltransferase. Toward this goal, frequencies measured in SAM's infrared spectra must be assigned to the motions of specific atoms via isotope incorporation at discrete positions. The incorporation of isotopes into SAM's structure can be accomplished via an established enzymatic synthesis using isotopically labeled precursors. However, published protocols produced an intense and highly variable IR signal which overlapped with many of the signals from SAM rendering comparison between isotopes challenging. We observed this intense absorption to be from co-purifying salts and the SAM counterion, producing a strong, broad signal at 1100 cm-1. Here, we report a revised SAM purification protocol that mitigates the contaminating salts and present the first IR spectra of isotopically labeled CD3-SAM. These results provide a foundation for isotopic labeling experiments of SAM that will define which atoms participate in individual molecular vibrations, as a means to detect specific molecular conformations.
Asunto(s)
Metionina , S-Adenosilmetionina , S-Adenosilmetionina/química , S-Adenosilmetionina/metabolismo , Sales (Química) , Metiltransferasas/química , Metiltransferasas/metabolismo , Racemetionina , IsótoposRESUMEN
This work demonstrates that chitin is an important structural component within the skeletal fibers of the freshwater sponge Spongilla lacustris. Using a variety of analytical techniques ((13)C solid state NMR, FT-IR, Raman, NEXAFS, ESI-MS, Morgan-Elson assay and Calcofluor White Staining); we show that this sponge chitin is much closer to α-chitin, known to be present in other animals, than to ß-chitin. Genetic analysis confirmed the presence of chitin synthases, which are described for the first time in a sponge. The presence of chitin in both marine (demosponges and hexactinellids) and freshwater sponges indicates that this important structural biopolymer was already present in their common ancestor.
Asunto(s)
Quitina/biosíntesis , Poríferos/metabolismo , Acetilglucosamina/metabolismo , Secuencias de Aminoácidos , Secuencia de Aminoácidos , Animales , Quitina/química , Quitina Sintasa/química , Quitina Sintasa/genética , Clonación Molecular , Datos de Secuencia Molecular , Poríferos/genética , Espectrometría de Masa por Ionización de Electrospray , Espectroscopía Infrarroja por Transformada de Fourier , Espectroscopía de Absorción de Rayos XRESUMEN
A holdfast is a root- or basal plate-like structure of principal importance that anchors aquatic sessile organisms, including sponges, to hard substrates. There is to date little information about the nature and origin of sponges' holdfasts in both marine and freshwater environments. This work, to our knowledge, demonstrates for the first time that chitin is an important structural component within holdfasts of the endemic freshwater demosponge Lubomirskia baicalensis. Using a variety of techniques (near-edge X-ray absorption fine structure, Raman, electrospray ionization mas spectrometry, Morgan-Elson assay and Calcofluor White staining), we show that chitin from the sponge holdfast is much closer to α-chitin than to ß-chitin. Most of the three-dimensional fibrous skeleton of this sponge consists of spicule-containing proteinaceous spongin. Intriguingly, the chitinous holdfast is not spongin-based, and is ontogenetically the oldest part of the sponge body. Sequencing revealed the presence of four previously undescribed genes encoding chitin synthases in the L. baicalensis sponge. This discovery of chitin within freshwater sponge holdfasts highlights the novel and specific functions of this biopolymer within these ancient sessile invertebrates.
Asunto(s)
Quitina Sintasa/genética , Quitina/química , Poríferos/química , Poríferos/genética , Acetilglucosamina/metabolismo , Secuencia de Aminoácidos , Animales , Bencenosulfonatos/metabolismo , Quitina/metabolismo , Quitina Sintasa/química , Quitina Sintasa/metabolismo , Medios de Contraste/metabolismo , Lagos , Datos de Secuencia Molecular , Filogenia , Reacción en Cadena de la Polimerasa , Poríferos/anatomía & histología , Federación de Rusia , Alineación de Secuencia , Espectrometría de Masa por Ionización de Electrospray , Espectrometría Raman , Espectroscopía de Absorción de Rayos XRESUMEN
The carbonyl stretching modes have been widely used in linear and two-dimensional infrared (IR) spectroscopy to probe the conformation, interaction, and biological functions of nucleic acids. However, due to their universal appearance in nucleobases, the IR absorption bands of nucleic acids are often highly congested in the 1600-1800 cm-1 region. Following the fruitful applications in proteins, 13C isotope labels have been introduced to the IR measurements of oligonucleotides to reveal their site-specific structural fluctuations and hydrogen bonding conditions. In this work, we combine recently developed frequency and coupling maps to develop a theoretical strategy that models the IR spectra of oligonucleotides with 13C labels directly from molecular dynamics simulations. We apply the theoretical method to nucleoside 5'-monophosphates and DNA double helices and demonstrate how elements of the vibrational Hamiltonian determine the spectral features and their changes upon isotope labeling. Using the double helices as examples, we show that the calculated IR spectra are in good agreement with experiments and the 13C isotope labeling technique can potentially be applied to characterize the stacking configurations and secondary structures of nucleic acids.
Asunto(s)
Oligonucleótidos , Proteínas , Espectrofotometría Infrarroja/métodos , Proteínas/química , ADN/química , Isótopos , NucleótidosRESUMEN
The level of interest in probing the strength of noncovalent interactions in DNA duplexes is high, as these weak forces dictate the range of suprastructures the double helix adopts under different conditions, in turn directly impacting the biological functions and industrial applications of duplexes that require making and breaking them to access the genetic code. However, few experimental tools can measure these weak forces embedded within large biological suprastructures in the native solution environment. Here, we develop experimental methods for detecting the presence of a single noncovalent interaction [a hydrogen bond (H-bond)] within a large DNA duplex in solution and measure its formation enthalpy (ΔHf). We report that introduction of a H-bond into the TC2âO group from the noncanonical nucleobase 2-aminopurine produces an expected decrease â¼10 ± 0.76 cm-1 (from â¼1720 cm-1 in Watson-Crick to â¼1710 cm-1 in 2-aminopurine), which correlates with an enthalpy of â¼0.93 ± 0.066 kcal/mol for this interaction.
Asunto(s)
2-Aminopurina , ADN , Temperatura , Conformación de Ácido Nucleico , Enlace de Hidrógeno , Termodinámica , ADN/química , Análisis EspectralRESUMEN
Structural bioinspiration in modern material science and biomimetics represents an actual trend that was originally based on the bioarchitectural diversity of invertebrate skeletons, specifically, honeycomb constructs of natural origin, which have been in humanities focus since ancient times. We conducted a study on the principles of bioarchitecture regarding the unique biosilica-based honeycomb-like skeleton of the deep-sea glass sponge Aphrocallistes beatrix. Experimental data show, with compelling evidence, the location of actin filaments within honeycomb-formed hierarchical siliceous walls. Principles of the unique hierarchical organization of such formations are discussed. Inspired by poriferan honeycomb biosilica, we designed diverse models, including 3D printing, using PLA-, resin-, and synthetic-glass-prepared corresponding microtomography-based 3D reconstruction.
RESUMEN
DNA helicase activity is essential for the vital DNA metabolic processes of recombination, replication, transcription, translation, and repair. Recently, an unexpected, rapid exponential ATP-stimulated DNA unwinding rate was observed from an Archaeoglobus fulgidus helicase (AfXPB) as compared to the slower conventional helicases from Sulfolobus tokodaii, StXPB1 and StXPB2. This unusual rapid activity suggests a "molecular wrench" mechanism arising from the torque applied by AfXPB on the duplex structure in transitioning from open to closed conformations. However, much remains to be understood. Here, we investigate the concentration dependence of DNA helicase binding and ATP-stimulated kinetics of StXPB2 and AfXPB, as well as their binding and activity in Bax1 complexes, via an electrochemical assay with redox-active DNA monolayers. StXPB2 ATP-stimulated activity is concentration-independent from 8 to 200 nM. Unexpectedly, AfXPB activity is concentration-dependent in this range, with exponential rate constants varying from seconds at concentrations greater than 20 nM to thousands of seconds at lower concentrations. At 20 nM, rapid exponential signal decay ensues, linearly reverses, and resumes with a slower exponential decay. This change in AfXPB activity as a function of its concentration is rationalized as the crossover between the fast molecular wrench and slower conventional helicase modes. AfXPB-Bax1 inhibits rapid activity, whereas the StXPB2-Bax1 complex induces rapid kinetics at higher concentrations. This activity is rationalized with the crystal structures of these complexes. These findings illuminate the different physical models governing molecular wrench activity for improved biological insight into a key factor in DNA repair.
Asunto(s)
Reparación del ADN , ADN , ADN/química , ADN Helicasas/química , Adenosina Trifosfato/metabolismo , CinéticaRESUMEN
Fourier transform infrared (FT-IR) spectroscopic imaging has been used to characterize different types of pituitary gland tumors and normal pituitary tissue. Freshly resected tumor tissue from surgery was prepared as thin cryosections and examined by FT-IR spectroscopic imaging. Tissue types were discriminated via k-means cluster analysis and a supervised classification algorithm based on linear discriminant analysis. Spectral classification allowed us to discriminate between tumor and non-tumor cells, as well as between tumor cells that produce human growth hormone (hGH+) and tumor cells that do not produce that hormone (hGH-). The spectral classification was compared and contrasted with a histological PAS and orange G stained image. It was further shown that hGH+ pituitary tumor cells show stronger amide bands than tumor cells that do not produce hGH. This study demonstrates that FT-IR spectroscopic imaging can not only potentially serve as a fast and objective approach for discriminating pituitary gland tumors from normal tissue, but that it can also detect hGH-producing tumor cells.
Asunto(s)
Hipófisis/patología , Neoplasias Hipofisarias/patología , Espectroscopía Infrarroja por Transformada de Fourier/métodos , Análisis por Conglomerados , Hormona de Crecimiento Humana/análisis , Humanos , Análisis Multivariante , Hipófisis/química , Neoplasias Hipofisarias/químicaRESUMEN
The blue light using flavin (BLUF) domain photosensors, such as the transcriptional antirepressor AppA, utilize a noncovalently bound flavin as the chromophore for photoreception. Since the isoalloxazine ring of the chromophore is unable to undergo large-scale structural change upon light absorption, there is intense interest in understanding how the BLUF protein matrix senses and responds to flavin photoexcitation. Light absorption is proposed to result in alterations in the hydrogen-bonding network that surrounds the flavin chromophore on an ultrafast time scale, and the structural changes caused by photoexcitation are being probed by vibrational spectroscopy. Here we report ultrafast time-resolved infrared spectra of the AppA BLUF domain (AppA(BLUF)) reconstituted with isotopically labeled riboflavin (Rf) and flavin adenine dinucleotide (FAD), which permit the first unambiguous assignment of ground and excited state modes arising directly from the flavin carbonyl groups. Studies of model compounds and DFT calculations of the ground state vibrational spectra reveal the sensitivity of these modes to their environment, indicating that they can be used as probes of structural dynamics.
Asunto(s)
Proteínas Bacterianas/química , Proteínas Bacterianas/metabolismo , Flavoproteínas/química , Flavoproteínas/metabolismo , Luz , Rhodobacter sphaeroides , Espectrofotometría Infrarroja/métodos , Flavina-Adenina Dinucleótido/metabolismo , Marcaje Isotópico , Cinética , Estructura Terciaria de Proteína , VibraciónRESUMEN
Photoexcitation of the flavin chromophore in the BLUF photosensor AppA results in a conformational change that leads to photosensor activation. This conformational change is mediated by a hydrogen-bonding network that surrounds the flavin, and photoexcitation is known to result in changes in the network that include a strengthening of hydrogen bonding to the flavin C4âO carbonyl group. Q63 is a key residue in the hydrogen-bonding network, and replacement of this residue with a glutamate results in a photoinactive mutant. While the ultrafast time-resolved infrared (TRIR) spectrum of Q63E AppA(BLUF) is characterized by flavin carbonyl modes at 1680 and 1650 cm(-1), which are similar in frequency to the analogous modes from the light activated state of the wild-type protein, a band is also observed in the TRIR spectrum at 1724 cm(-1) that is unambiguously assigned to the Q63E carboxylic acid based on U-(13)C labeling of the protein. Light absorption instantaneously (<100 fs) bleaches the 1724 cm(-1) band leading to a transient absorption at 1707 cm(-1). Because Q63E is not part of the isoalloxazine electronic transition, the shift in frequency must arise from a sub picosecond perturbation to the flavin binding pocket. The light-induced change in the frequency of the Q63E side chain is assigned to an increase in hydrogen-bond strength of 3 kcal mol(-1) caused by electronic reorganization of the isoalloxazine ring in the excited state, providing direct evidence that the protein matrix of AppA responds instantaneously to changes in the electronic structure of the chromophore and supporting a model for photoactivation of the wild-type protein that involves initial tautomerization of the Q63 side chain.
Asunto(s)
Flavina-Adenina Dinucleótido/química , Luz , Células Fotorreceptoras/química , Enlace de Hidrógeno , Estructura Molecular , Espectrofotometría Infrarroja , Espectrometría RamanRESUMEN
Established methods for characterization of tissue and diagnostics, for example histochemistry, magnetic resonance imaging (MRI), X-ray tomography, or positron emission tomography (PET), are mostly not suitable for intra-operative use. However, there is a clear need for an intra-operative diagnostics especially to identify the borderline between normal and tumor tissue. Currently, vibrational spectroscopy techniques (both Raman and infrared) complement the standard methods for tissue diagnostics. Vibrational spectroscopy has the potential for intra-operative use, because it can provide a biochemically based profile of tissue in real time and without requiring additional contrast agents, which may perturb the tissue under investigation. In addition, no electric potential needs to be applied, and the measurements are not affected by electromagnetic fields. Currently, promising approaches include Raman fiber techniques and nonlinear Raman spectroscopy. Infrared spectroscopy is also being used to examine freshly resected tissue ex vivo in the operating theater. The immense volume of information contained in Raman and infrared spectra requires multivariate analysis to extract relevant information to distinguish different types of tissue. The promise and limitations of vibrational spectroscopy methods as intra-operative tools are surveyed in this review.
Asunto(s)
Espectrofotometría Infrarroja/métodos , Espectrometría Raman/métodos , Animales , Humanos , Procedimientos Quirúrgicos Operativos/métodosRESUMEN
Each year, billions of day-old layer chicks are produced in the world. Since only female chicks are reared for egg production, the chicks must be sexed and the unwanted male layer chicks are culled. The culling of male chicks is a serious problem, both in terms of animal welfare and waste disposal. The germinal disc in fertilized but unincubated eggs contains already several thousands of blastoderm cells. The cellular DNA in birds is different for male and female chicks. The difference in DNA content between male and female chicks is around 2% and is measurable by Fourier transform infrared (FT-IR) spectroscopy. In this study, small amounts of blastoderm cells from 22 chicken eggs were characterized by attenuated total reflection FT-IR spectroscopic imaging and classified by linear discriminant analysis. Polymerase chain reaction (PCR) was used as a reference method to determine the gender. The spectroscopic results demonstrate that male blastoderm cells exhibit a higher content of DNA than cells from female blastoderm. The spectroscopic-based gender determination led to the same result as the PCR analysis. FT-IR spectroscopic imaging allows the gender determination of unincubated eggs within a few seconds based on the accurate determination of the different DNA contents in blastoderm cells of both sexes.