RESUMEN
Photosynthetic reaction centres harvest the energy content of sunlight by transporting electrons across an energy-transducing biological membrane. Here we use time-resolved serial femtosecond crystallography1 using an X-ray free-electron laser2 to observe light-induced structural changes in the photosynthetic reaction centre of Blastochloris viridis on a timescale of picoseconds. Structural perturbations first occur at the special pair of chlorophyll molecules of the photosynthetic reaction centre that are photo-oxidized by light. Electron transfer to the menaquinone acceptor on the opposite side of the membrane induces a movement of this cofactor together with lower amplitude protein rearrangements. These observations reveal how proteins use conformational dynamics to stabilize the charge-separation steps of electron-transfer reactions.
Asunto(s)
Proteínas del Complejo del Centro de Reacción Fotosintética/química , Proteínas del Complejo del Centro de Reacción Fotosintética/metabolismo , Bacterioclorofilas/metabolismo , Sitios de Unión/efectos de los fármacos , Clorofila/metabolismo , Clorofila/efectos de la radiación , Cristalografía , Citoplasma/metabolismo , Transporte de Electrón/efectos de los fármacos , Electrones , Hyphomicrobiaceae/enzimología , Hyphomicrobiaceae/metabolismo , Rayos Láser , Modelos Moleculares , Oxidación-Reducción/efectos de la radiación , Feofitinas/metabolismo , Proteínas del Complejo del Centro de Reacción Fotosintética/efectos de la radiación , Protones , Ubiquinona/análogos & derivados , Ubiquinona/metabolismo , Vitamina K 2/metabolismoRESUMEN
Preparation of cryoelectron microscopy (cryo-EM) grids for imaging of amyloid fibrils is notoriously challenging. The human islet amyloid polypeptide (hIAPP) serves as a notable example, as the majority of reported structures have relied on the use of nonphysiological pH buffers, N-terminal tags, and seeding. This highlights the need for more efficient, reproducible methodologies that can elucidate amyloid fibril structures formed under diverse conditions. In this work, we demonstrate that the distribution of fibrils on cryo-EM grids is predominantly determined by the solution composition, which is critical for the stability of thin vitreous ice films. We discover that, among physiological pH buffers, HEPES uniquely enhances the distribution of fibrils on cryo-EM grids and improves the stability of ice layers. This improvement is attributed to direct interactions between HEPES molecules and hIAPP, effectively minimizing the tendency of hIAPP to form dense clusters in solutions and preventing ice nucleation. Furthermore, we provide additional support for the idea that denatured protein monolayers forming at the interface are also capable of eliciting a surfactant-like effect, leading to improved particle coverage. This phenomenon is illustrated by the addition of nonamyloidogenic rat IAPP (rIAPP) to a solution of preaggregated hIAPP just before the freezing process. The resultant grids, supplemented with this "spectator protein", exhibit notably enhanced coverage and improved ice quality. Unlike conventional surfactants, rIAPP is additionally capable of disentangling the dense clusters formed by hIAPP. By applying the proposed strategies, we have resolved the structure of the dominant hIAPP polymorph, formed in vitro at pH 7.4, to a final resolution of 4 Å. The advances in grid preparation presented in this work hold significant promise for enabling structural determination of amyloid proteins which are particularly resistant to conventional grid preparation techniques.
Asunto(s)
Amiloide , Hielo , Ratas , Animales , Humanos , Amiloide/química , Microscopía por Crioelectrón , HEPES , Polipéptido Amiloide de los Islotes Pancreáticos/químicaRESUMEN
In order to use the infrared (IR) radiation shielding materials, they should take a form of thin film coatings deposited on glass/polymer substrates or be used as fillers of glass/polymer. The first approach usually suffers from several technological problems. Therefore, the second strategy gains more and more attention. Taking into account this trend, this work presents the usage of iron nanoparticles (Fe NPs) embedded into the poly(vinylidene fluoride-co-hexafluoropropylene) (PVDF-HFP) films as the shielding material in near-infrared (NIR) and mid-infrared (MIR) region. The performed investigations show that the transmittance of copolymer films decreases with increasing content of the Fe NPs inside them. It is found that the average fade of IR transmittance for 1, 2.5, 5, 10, and 50 mg of Fe NPs is about 13%, 24%, 31%, 77%, and 98%, respectively. Moreover, it is observed that the PVDF-HFP films filled in the Fe NPs almost does not reflect the NIR and MIR radiation. Hence, the IR shielding properties of the PVDF-HFP films can be effectively tuned by the addition of proper amount of the Fe NPs. This, in turn, shows that the PVDF-HFP films filled in the Fe NPs constitute a great option for IR antireflective and shielding applications.
Asunto(s)
Hierro , Nanopartículas , Polivinilos/química , Polímeros , Nanopartículas/químicaRESUMEN
Phytochromes belong to a group of photoreceptor proteins containing a covalently bound biliverdin chromophore that inter-converts between two isomeric forms upon photoexcitation. The existence and stability of the photocycle products are largely determined by the protein sequence and the presence of conserved hydrogen-bonding interactions in the vicinity of the chromophore. The vibrational signatures of biliverdin, however, are often weak and obscured under more intense protein bands, limiting spectroscopic studies of its non-transient signals. In this study, we apply isotope-labeling techniques to isolate the vibrational bands from the protein-bound chromophore of the bacterial phytochrome from Deinococcus radiodurans. We elucidate the structure and ultrafast dynamics of the chromophore with 2D infra-red (IR) spectroscopy and molecular dynamics simulations. The carbonyl stretch vibrations of the pyrrole rings show the heterogeneous distribution of hydrogen-bonding structures, which exhibit distinct ultrafast relaxation dynamics. Moreover, we resolve a previously undetected 1678 cm-1 band that is strongly coupled to the A- and D-ring of biliverdin and demonstrate the presence of complex vibrational redistribution pathways between the biliverdin modes with relaxation-assisted measurements of 2D IR cross peaks. In summary, we expect 2D IR spectroscopy to be useful in explaining how point mutations in the protein sequence affect the hydrogen-bonding structure around the chromophore and consequently its ability to photoisomerize to the light-activated states.
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Biliverdina , Fitocromo , Vibración , Espectrofotometría Infrarroja , HidrógenoRESUMEN
This paper presents a novel, autonomous learning system working in real-time for face recognition. Multiple convolutional neural networks for face recognition tasks are available; however, these networks need training data and a relatively long training process as the training speed depends on hardware characteristics. Pretrained convolutional neural networks could be useful for encoding face images (after classifier layers are removed). This system uses a pretrained ResNet50 model to encode face images from a camera and the Multinomial Naïve Bayes for autonomous training in the real-time classification of persons. Faces of several persons visible in a camera are tracked using special cognitive tracking agents who deal with machine learning models. After a face in a new position of the frame appears (in a place where there was no face in the previous frames), the system checks if it is novel or not using a novelty detection algorithm based on an SVM classifier; if it is unknown, the system automatically starts training. As a result of the conducted experiments, one can conclude that good conditions provide assurance that the system can learn the faces of a new person who appears in the frame correctly. Based on our research, we can conclude that the critical element of this system working is the novelty detection algorithm. If false novelty detection works, the system can assign two or more different identities or classify a new person into one of the existing groups.
Asunto(s)
Algoritmos , Redes Neurales de la Computación , Humanos , Teorema de Bayes , Aprendizaje AutomáticoRESUMEN
Phytochromes are photosensory proteins in plants, fungi, and bacteria, which detect red- and far-red light. They undergo a transition between the resting (Pr) and photoactivated (Pfr) states. In bacterial phytochromes, the Pr-to-Pfr transition is facilitated by two intermediate states, called Lumi-R and Meta-R. The molecular structures of the protein in these states are not known and the molecular mechanism of photoconversion is not understood. Here, we apply transient infrared absorption spectroscopy to study the photocycle of the wild-type and Y263F mutant of the phytochrome from Deinococcus radiodurans (DrBphP) from nano- to milliseconds. We identify two sequentially forming Lumi-R states which differ in the local structure surrounding the carbonyl group of the biliverdin D-ring. We also find that the tyrosine at position 263 alters local structure and dynamics around the D-ring and causes an increased rate of Pfr formation. The results shed new light on the mechanism of light-signalling in phytochrome proteins.
Asunto(s)
Deinococcus/química , Deinococcus/genética , Modelos Moleculares , Fitocromo/química , Espectrofotometría Infrarroja , Proteínas Bacterianas/química , Fototransducción/genética , Mutación , Estructura Terciaria de ProteínaRESUMEN
It is now recognized that many amyloid-forming proteins can associate into multiple fibril structures. Here, we use two-dimensional infrared spectroscopy to study two fibril polymorphs formed by human islet amyloid polypeptide (hIAPP or amylin), which is associated with type 2 diabetes. The polymorphs exhibit different degrees of structural organization near the loop region of hIAPP fibrils. The relative populations of these polymorphs are systematically altered by the presence of macrocyclic peptides which template ß-sheet formation at specific sections of the hIAPP sequence. These experiments are consistent with polymorphs that result from competing pathways for fibril formation and that the macrocycles bias hIAPP aggregation toward one pathway or the other. Another macrocyclic peptide that matches the loop region but extends the lag time leaves the relative populations of the polymorphs unaltered, suggesting that the branching point for structural divergence occurs after the lag phase, when the oligomers convert into seeds that template fibril formation. Thus, we conclude that the structures of the polymorphs stem from restricting oligomers along diverging folding pathways, which has implications for drug inhibition, cytotoxicity, and the free energy landscape of hIAPP aggregation.
Asunto(s)
Agonistas de los Receptores de Amilina/química , Amiloide/química , Polipéptido Amiloide de los Islotes Pancreáticos/química , Conformación Proteica , Humanos , Espectrofotometría InfrarrojaRESUMEN
The interplay between the intracellular gate and the selectivity filter underlies the structural basis for gating in potassium ion channels. Using a combination of protein semisynthesis, two-dimensional infrared (2D IR) spectroscopy, and molecular dynamics (MD) simulations, we probe the ion occupancy at the S1 binding site in the constricted state of the selectivity filter of the KcsA channel when the intracellular gate is open and closed. The 2D IR spectra resolve two features, whose relative intensities depend on the state of the intracellular gate. By matching the experiment to calculated 2D IR spectra of structures predicted by MD simulations, we identify the two features as corresponding to states with S1 occupied or unoccupied by K+. We learn that S1 is >70% occupied when the intracellular gate is closed and <15% occupied when the gate is open. Comparison of MD trajectories show that opening of the intracellular gate causes a structural change in the selectivity filter, which leads to a change in the ion occupancy. This work reveals the complexity of the conformational landscape of the K+ channel selectivity filter and its dependence on the state of the intracellular gate.
Asunto(s)
Activación del Canal Iónico , Simulación de Dinámica Molecular , Canales de Potasio/química , Sitios de Unión , Espectrofotometría InfrarrojaRESUMEN
Conformational changes are central to protein function but challenging to characterize with both high spatial and temporal precision. The inherently fast time scale and small chromophores of infrared (IR) spectroscopy are well-suited for characterization of potentially rapidly fluctuating environments, and when frequency-resolved probes are incorporated to overcome spectral congestion, enable characterization of specific sites in proteins. We selectively incorporated p-cyanophenylalanine (CNF) as a vibrational probe at five distinct locations in the enzyme cytochrome P450cam and used IR spectroscopy to characterize the environments in substrate and/or ligand complexes reflecting those in the catalytic cycle. Molecular dynamics (MD) simulations were performed to provide a structural basis for spectral interpretation. Together the experimental and simulation data suggest that the CN frequencies are sensitive to both long-range influences, resulting from the particular location of a residue within the enzyme, as well as short-range influences from hydrogen bonding and packing interactions. The IR spectra demonstrate that the environments and effects of substrate and/or ligand binding are different at each position probed and also provide evidence that a single site can experience multiple environments. This study illustrates how IR spectroscopy, when combined with the spectral decongestion and spatial selectivity afforded by CNF incorporation, provides detailed information about protein structural changes that underlie function.
Asunto(s)
Alcanfor 5-Monooxigenasa/química , Pseudomonas putida/enzimología , Espectroscopía Infrarroja por Transformada de Fourier/métodos , Alanina/análogos & derivados , Alanina/química , Alanina/metabolismo , Alcanfor/metabolismo , Alcanfor 5-Monooxigenasa/metabolismo , Simulación de Dinámica Molecular , Nitrilos/química , Nitrilos/metabolismo , Unión Proteica , Conformación Proteica , Pseudomonas putida/química , Pseudomonas putida/metabolismoRESUMEN
Structural dynamics within the distal cavity of myoglobin protein is investigated using 2D-IR and IR pump-probe spectroscopy of the N≡C stretch modes of heme-bound thiocyanate and selenocyanate ions. Although myoglobin-bound thiocyanate group shows a doublet in its IR absorption spectrum, no cross peaks originating from chemical exchange between the two components are observed in the time-resolved 2D IR spectra within the experimental time window. Frequency-frequency correlation functions of the two studied anionic ligands are obtained by means of a few different analysis approaches; these functions were then used to elucidate the differences in structural fluctuation around ligand, ligand-protein interactions, and the degree of structural heterogeneity within the hydrophobic pocket of these myoglobin complexes.
Asunto(s)
Cianatos/química , Mioglobina/química , Compuestos de Selenio/química , Tiocianatos/química , Animales , Cianatos/metabolismo , Caballos , Iones/química , Mioglobina/metabolismo , Compuestos de Selenio/metabolismo , Espectroscopía Infrarroja por Transformada de Fourier , Tiocianatos/metabolismoRESUMEN
An infrared (IR) probe based on isonitrile (NC)-derivatized alanine 1 was synthesized and the vibrational properties of its NC stretching mode were investigated using FTIR and femtosecond IR pump-probe spectroscopy. It is found that the NC stretching mode is very sensitive to the hydrogen-bonding ability of solvent molecules. Moreover, its transition dipole strength is larger than that of nitrile (CN) in nitrile-derivatized IR probe 2. The vibrational lifetime of the NC stretching mode is found to be 5.5 ± 0.2 ps in both D2O and DMF solvents, which is several times longer than that of the azido (N3) stretching mode in azido-derivatized IR probe 3. Altogether these properties suggest that the NC group can be a very promising sensing moiety of IR probes for studying the solvation structure and dynamics of biomolecules.
Asunto(s)
Alanina/análogos & derivados , Alanina/química , Rayos Infrarrojos , Sondas Moleculares/química , Nitrilos/química , Vibración , Conformación Molecular , Espectrofotometría InfrarrojaRESUMEN
The structure and vibrational dynamics of SCN- and SeCN-bound myoglobin have been investigated using polarization-controlled IR pump-probe measurements and quantum chemistry calculations. The complexes are found to be in low and high spin states, with the dominant contribution from the latter. In addition, the Mb:SCN high spin complex exhibits a doublet feature in the thiocyanate stretch IR absorption spectra, indicating two distinct molecular conformations around the heme pocket. The binding mode of the high spin complexes was assigned to occur through the nitrogen atom, contrary to the binding through the sulfur atom that was observed in myoglobin derived from Aplysia Limacina. The vibrational energy relaxation process has been found to occur substantially faster than those of free SCN(-) and SeCN(-) ions and neutral SCN- and SeCN-derivatized molecules reported previously. This supports the N-bound configurations of MbNCS and MbNCSe, because S- and Se-bound configurations are expected to have significantly long lifetimes due to the insulation effect by heavy bridge atom like S and Se in such IR probes. Nonetheless, even though their lifetimes are much shorter than those of corresponding free ions in water, the vibrational lifetimes determined for MbNCS and MbNCSe are still fairly long compared to those of azide and cyanide myoglobin systems studied before. Thus, thiocyanate and selenocyanate can be good local probes of local electrostatic environment in the heme pocket. The globin dependence on binding mode and vibrational dynamics is also discussed.
Asunto(s)
Cianatos/química , Mioglobina/química , Compuestos de Selenio/química , Tiocianatos/química , Animales , Caballos , Conformación Molecular , Miocardio/química , Espectroscopía Infrarroja por Transformada de Fourier , Vibración , Agua/químicaRESUMEN
Charge-transfer reactions in proteins are important for life, such as in photolyases which repair DNA, but the role of structural dynamics remains unclear. Here, using femtosecond X-ray crystallography, we report the structural changes that take place while electrons transfer along a chain of four conserved tryptophans in the Drosophila melanogaster (6-4) photolyase. At femto- and picosecond delays, photoreduction of the flavin by the first tryptophan causes directed structural responses at a key asparagine, at a conserved salt bridge, and by rearrangements of nearby water molecules. We detect charge-induced structural changes close to the second tryptophan from 1 ps to 20 ps, identifying a nearby methionine as an active participant in the redox chain, and from 20 ps around the fourth tryptophan. The photolyase undergoes highly directed and carefully timed adaptations of its structure. This questions the validity of the linear solvent response approximation in Marcus theory and indicates that evolution has optimized fast protein fluctuations for optimal charge transfer.
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Desoxirribodipirimidina Fotoliasa , Humanos , Animales , Desoxirribodipirimidina Fotoliasa/química , Desoxirribodipirimidina Fotoliasa/genética , Desoxirribodipirimidina Fotoliasa/metabolismo , Triptófano/química , Electrones , Drosophila melanogaster/metabolismo , Escherichia coli/genética , Transporte de Electrón , Cristalografía por Rayos XRESUMEN
The origins of stabilization in the short strong hydrogen bonds commonly referred to as "resonance-assisted" (RAHB) have been revisited using the modern valence-bond theory, the hybrid variational-perturbational interaction energy decomposition scheme and atoms in molecules (AIM) analysis. Dimers of carboxylic acids and amides have been chosen as the model structures for intermolecular RAHBs, while for the intramolecular case malondialdehyde and its substituted derivatives have been selected. The estimated (negligible) resonance stabilization energies and relative magnitudes of interaction energy components indicate that the origin of stabilization in the studied complexes is charge-delocalization. Although in the case of intramolecular RAHBs the resonance effects are much more pronounced, still they are a relatively minor contribution to the total stabilization energy. In fact, the estimated resonance stabilization energies diminish with an increasing strength of the hydrogen bond (as indicated by AIM and structural descriptors).
RESUMEN
Certain cyanine dye molecules have been observed to self-assemble in DNA templates to form large chiral aggregates, which exhibit induced circular dichroism. The structure and circular dichroism (CD) of one such system, aggregates of a cationic DiSC2(5) cyanine dye, are investigated using the time-dependent Kohn-Sham density functional theory (TD-DFT) and exciton coupling model. A series of TD-DFT calculations on the aggregates with one, two, and four dye molecules clearly shows the onset of CD induced by the helically twisted structure compatible with the minor groove of DNA templates. More simplified exciton coupling model analysis successfully reproduces the major positive Cotton effect observed in the experiment as well as the TD-DFT calculations, but it is unable to capture minor features of the CD spectrum that are closely related to absolute configurations of constituent dyes in the complex. We assess the performance of various methods used for evaluation of the electronic coupling energies between interacting chromophores. Our results confirm that the interchromophore interactions in cyanine dye aggregates are primarily electrostatic in nature and indicate that the exciton coupling model is adequate for studying induced CD of self-assembled aggregates of cyanine dye molecules.
Asunto(s)
Colorantes/química , ADN/química , Ditiazanina/química , Fenómenos Ópticos , Teoría Cuántica , Simulación de Dinámica Molecular , Conformación de Ácido NucleicoRESUMEN
Long-term functional storage of therapeutic proteins at room temperature has been an eternal challenge. Inspired by the cellular cooperativity of proteins, we have taken a step forward to address this challenge by cohabitating Immunoglobulin G (IgG1) with a food protein gelatin in the solid-state at room temperature. Interestingly, IgG1 remained functionally active for a record 14 months revealed from the western-blot assay. Further quantification by HP-LC analysis showed 100% structural integrity of IgG1 with no degradation in the gelatin matrix during this period. The developed formulation has a direct application in oral medical nutrition therapy to cure gastrointestinal microbial infections. Also the strategy provides a robust energy economic alternative to the protein engineering methods for long-term functional storage of therapeutic proteins at room temperature.
Asunto(s)
Gelatina , Inmunoglobulina G , Inmunoglobulina G/química , TemperaturaRESUMEN
The aggregation of human islet amyloid polypeptide (hIAPP) into amyloid fibrils involves formation of oligomeric intermediates that are thought to be the cytotoxic species responsible for ß-cell dysfunction in type 2 diabetes. hIAPP oligomers permeating or disrupting the cellular membrane may be one mechanism of toxicity and so measuring the structural kinetics of aggregation in the presence of membranes is of much interest. In this study, we use 2D IR spectroscopy and 13C18O isotope labeling to study the secondary structure of the oligomeric intermediates formed in solution and in the presence of phospholipid vesicles at sites L12A13, L16V17, G24A25 and V32G33. Pairs of labels monitor the couplings between associated polypeptides and the dihedral angles between adjacent residues. In solution, the L12A13 residues form an oligomeric ß-sheet in addition to an α-helix whereas with the phospholipid vesicles they are α-helical throughout the aggregation process. In both solution and with DOPC vesicles, L16V17 and V32G33 have disordered structures until fibrils are formed. Similarly, under both conditions, G24A25 exhibits 3-state kinetics, created by an oligomeric intermediate with a well-defined ß-sheet structure. Amyloid fibril formation is often thought to involve intermediates with exceedingly low populations that are difficult to detect experimentally. These experiments establish that amyloid fibril formation of hIAPP when catalyzed by membranes includes a metastable intermediate and that this intermediate has a similar structure at G24A25 in the FGAIL region as the corresponding intermediate in solution, thought to be the toxic species.
RESUMEN
Phytochromes are red/far-red light photoreceptors in bacteria to plants, which elicit a variety of important physiological responses. They display a reversible photocycle between the resting Pr state and the light-activated Pfr state. Light signals are transduced as structural change through the entire protein to modulate its activity. It is unknown how the Pr-to-Pfr interconversion occurs, as the structure of intermediates remains notoriously elusive. Here, we present short-lived crystal structures of the photosensory core modules of the bacteriophytochrome from myxobacterium Stigmatella aurantiaca captured by an X-ray free electron laser 5 ns and 33 ms after light illumination of the Pr state. We observe large structural displacements of the covalently bound bilin chromophore, which trigger a bifurcated signaling pathway that extends through the entire protein. The snapshots show with atomic precision how the signal progresses from the chromophore, explaining how plants, bacteria, and fungi sense red light.
Asunto(s)
Fitocromo/química , Fitocromo/metabolismo , Stigmatella aurantiaca/metabolismo , Proteínas Bacterianas/química , Proteínas Bacterianas/metabolismo , Sitios de Unión , Cristalografía por Rayos X , Modelos Moleculares , Conformación ProteicaRESUMEN
[This corrects the article DOI: 10.1155/2018/2827580.].