RESUMO
The SARS-CoV-2 virus is highly contagious, as demonstrated by numerous well-documented superspreading events. The infection commonly starts in the upper respiratory tract (URT) but can migrate to the lower respiratory tract (LRT) and other organs, often with severe consequences. Whereas LRT infection can lead to shedding of virus via breath and cough droplets, URT infection enables shedding via abundant speech droplets. Their viral load can be high in carriers with mild or no symptoms, an observation linked to the abundance of SARS-CoV-2-susceptible cells in the oral cavity epithelium. Expelled droplets rapidly lose water through evaporation, with the smaller ones transforming into long-lived aerosol. Although the largest speech droplets can carry more virions, they are few in number, fall to the ground rapidly and therefore play a relatively minor role in transmission. Of more concern is small speech aerosol, which can descend deep into the LRT and cause severe disease. However, since their total volume is small, the amount of virus they carry is low. Nevertheless, in closed environments with inadequate ventilation, they can accumulate, which elevates the risk of direct LRT infection. Of most concern is the large fraction of speech aerosol that is intermediate-sized because it remains suspended in air for minutes and can be transported over considerable distances by convective air currents. The abundance of this speech-generated aerosol, combined with its high viral load in pre- and asymptomatic individuals, strongly implicates airborne transmission of SARS-CoV-2 through speech as the primary contributor to its rapid spread.
Assuntos
COVID-19/epidemiologia , Tosse/etiologia , Transmissão de Doença Infecciosa/estatística & dados numéricos , SARS-CoV-2/genética , Espirro , Carga Viral , COVID-19/complicações , Saúde Global , Humanos , PandemiasRESUMO
BioCARS, a NIH-supported national user facility for macromolecular time-resolved X-ray crystallography at the Advanced Photon Source (APS), has recently completed commissioning of an upgraded undulator-based beamline optimized for single-shot laser-pump X-ray-probe measurements with time resolution as short as 100 ps. The source consists of two in-line undulators with periods of 23 and 27 mm that together provide high-flux pink-beam capability at 12 keV as well as first-harmonic coverage from 6.8 to 19 keV. A high-heat-load chopper reduces the average power load on downstream components, thereby preserving the surface figure of a Kirkpatrick-Baez mirror system capable of focusing the X-ray beam to a spot size of 90 µm horizontal by 20 µm vertical. A high-speed chopper isolates single X-ray pulses at 1 kHz in both hybrid and 24-bunch modes of the APS storage ring. In hybrid mode each isolated X-ray pulse delivers up to ~4 × 10(10) photons to the sample, thereby achieving a time-averaged flux approaching that of fourth-generation X-FEL sources. A new high-power picosecond laser system delivers pulses tunable over the wavelength range 450-2000 nm. These pulses are synchronized to the storage-ring RF clock with long-term stability better than 10 ps RMS. Monochromatic experimental capability with Biosafety Level 3 certification has been retained.
Assuntos
Síncrotrons , Cristalografia por Raios XRESUMO
The relative orientations of carbon monoxide (CO) bound to and photodissociated from myoglobin in solution have been determined with time-resolved infrared polarization spectroscopy. The bound CO is oriented < or = 7 degrees from the heme normal, corresponding to nearly linear FE-C-O. Upon dissociation from the Fe, CO becomes trapped in a docking site that orientationally constrains it to lie approximately in the plane of the heme. Because the bound and "docked" CO are oriented in nearly orthogonal directions CO binding from the docking site is suppressed. These solutions results help to establish how myoglobin discriminates against CO, a controversial issue dominated by the misconception that Fe-C-O is bent.
Assuntos
Monóxido de Carbono/química , Mioglobina/química , Sítios de Ligação , Monóxido de Carbono/metabolismo , Cristalografia por Raios X , Ligantes , Luz , Mioglobina/metabolismo , Oxigênio/química , Oxigênio/metabolismo , Fotólise , Conformação Proteica , Espectrofotometria Infravermelho , TemperaturaRESUMO
Chemical dynamics in proteins are discussed, with bacteriorhodopsin serving as a model system. Ultrafast time-resolved methods used to probe the chemical dynamics of retinal photoisomerization in bacteriorhodopsin are discussed, along with future prospects for ultrafast time-resolved crystallography. The photoisomerization of retinal in bacteriorhodopsin is far more selective and efficient than in solution, the origins of which are discussed in the context of a three-state model for the photoisomerization reaction coordinate. The chemical dynamics are complex, with the excited-state relaxation exhibiting a multiexponential decay with well-defined rate constants. Possible origins for the two major components are also discussed.
Assuntos
Bacteriorodopsinas/química , Luz , Retinaldeído/química , Bacteriorodopsinas/metabolismo , Diterpenos , Halobacterium salinarum/química , Halobacterium salinarum/metabolismo , Isomerismo , Cinética , Modelos Químicos , Conformação Proteica , Retinaldeído/metabolismo , Análise Espectral , TermodinâmicaRESUMO
Recent developments in time-resolved infrared spectroscopy have paved the way to probe transient intermediates with a high degree of functional group specificity on timescales as short as femtoseconds. This capability has been exploited in studies of biophysical phenomena ranging from protein folding/unfolding to ligand migration in proteins.
Assuntos
Conformação Proteica , Dobramento de Proteína , Proteínas/química , Espectrofotometria Infravermelho/métodos , Proteínas de Bactérias/química , Complexo IV da Cadeia de Transporte de Elétrons/química , Fotossíntese , Proteínas/metabolismo , Fatores de TempoRESUMO
We present a crystallography chip enabling in situ room temperature crystallography at microfocus synchrotron beamlines and X-ray free-electron laser (X-FEL) sources. Compared to other in situ approaches, we observe extremely low background and high diffraction data quality. The chip design is robust and allows fast and efficient loading of thousands of small crystals. The ability to load a large number of protein crystals, at room temperature and with high efficiency, into prescribed positions enables high throughput automated serial crystallography with microfocus synchrotron beamlines. In addition, we demonstrate the application of this chip for femtosecond time-resolved serial crystallography at the Linac Coherent Light Source (LCLS, Menlo Park, California, USA). The chip concept enables multiple images to be acquired from each crystal, allowing differential detection of changes in diffraction intensities in order to obtain high signal-to-noise and fully exploit the time resolution capabilities of XFELs.
RESUMO
The picosecond evolution of the tertiary conformation of myoglobin (Mb) after photodissociation of MbCO was investigated at room temperature by probing band III, a weak iron-porphyrin charge-transfer transition near 13,110 cm-1 (763 nm) that is sensitive to the out-of-plane displacement of the iron. Upon photolysis, the iron moves out of the plane of the porphyrin, causing a blue-shift of band III and a concomitant change in the protein conformation. The dynamics for this functionally important motion are highly nonexponential, in agreement with recent molecular dynamics simulations [Kuczera, K., Lambry, J.-C., Martin, J.-L. & Karplus, M. (1993) Proc. Natl. Acad. Sci. USA 90, 5805-5807]. The conformational change likely affects the height of the barrier to ligand rebinding and may explain nonexponential NO rebinding.
Assuntos
Mioglobina/química , Conformação Proteica , Estrutura Terciária de Proteína , Dicroísmo Circular , Cinética , Lasers , Fotólise , Fatores de TempoRESUMO
The photodissociation of CO from HbCO at ambient temperature is studied by means of a femtosecond IR technique. The bleaching of the FeCO absorption and the appearance of a new IR absorption near that of free CO are both observed at 300 fs after optical excitation. The bleach does not recover on the time scale of a few picoseconds but does recover by approximately 4% within 1 ns, which suggests that a barrier to recombination is formed within a few picoseconds. The CO spectrum does not change significantly between 300 fs and 1 ns, suggesting that the CO quickly finds some locations in the heme pocket that are not more than a few angstroms from the iron. The de-ligated CO appears in its ground vibrational level. There is evidence that 85 +/- 10% of this CO remains in the heme pocket at 1 ns; it probably resides there for 50 ns. The flow of excess vibrational energy from the heme to the solvent was directly observed in the IR experiments. The heme cools within 1-2 ps while thermal disruption of the surrounding solvent structure requires approximately 30 ps.
Assuntos
Carboxihemoglobina/metabolismo , Hemoglobinas/metabolismo , Deutério , Óxido de Deutério , Humanos , Cinética , Lasers , Ligantes , Fotólise , Espectrofotometria Infravermelho/métodos , Fatores de Tempo , ÁguaRESUMO
The nature of ligand motion within proteins has been investigated by measuring femtosecond time-resolved infrared (IR) spectra of CO photodissociated from the haem of myoglobin. Upon dissociation, the CO rotates approximately 90 degrees and becomes trapped within a ligand docking site located near the binding site. Two trajectories, distinguished spectroscopically and kinetically with time constants of 0.20 +/- 0.05 ps and 0.52 +/- 0.10 ps, lead to CO located within the docking site with opposite orientations. The protein reorganizes about the "docked' CO with a time constant of 1.6 +/- 0.3 ps and quickly establishes an energetic barrier that inhibits the reverse rebinding process.
Assuntos
Dióxido de Carbono/metabolismo , Mioglobina/química , Conformação Proteica , Animais , Cavalos , Cinética , Ligantes , Modelos Químicos , Mioglobina/metabolismo , Rotação , Espectrofotometria Infravermelho , Fatores de TempoRESUMO
The primary events in the all-trans to 13-cis photoisomerization of retinal in bacteriorhodopsin have been investigated with femtosecond time-resolved absorbance spectroscopy. Spectra measured over a broad range extending from 7000 to 22,400 cm-1 reveal features whose dynamics are inconsistent with a model proposed earlier to account for the highly efficient photoisomerization process. Emerging from this work is a new three-state model. Photoexcitation of retinal with visible light accesses a shallow well on the excited state potential energy surface. This well is bounded by a small barrier, arising from an avoided crossing that separates the Franck-Condon region from the nearby reactive region of the photoisomerization coordinate. At ambient temperatures, the reactive region is accessed with a time constant of approximately 500 fs, whereupon the retinal rapidly twists and encounters a second avoided crossing region. The protein mediates the passage into the second avoided crossing region and thereby exerts control over the quantum yield for forming 13-cis retinal. The driving force for photoisomerization resides in the retinal, not in the surrounding protein. This view contrasts with an earlier model where photoexcitation was thought to access directly a reactive region of the excited-state potential and thereby drive the retinal to a twisted conformation within 100-200 fs.
Assuntos
Bacteriorodopsinas/química , Retinaldeído/química , Bacteriorodopsinas/efeitos da radiação , Isomerismo , Cinética , Modelos Químicos , Fotoquímica , Retinaldeído/efeitos da radiação , Termodinâmica , Fatores de TempoRESUMO
Many elementary chemical and physical processes such as the breaking of a chemical bond or the vibrational motion of atoms within a molecule take place on a femtosecond (fs = 10(-15)s) or picosecond (ps = 10(-12)s) time scale. It is now possible to monitor these events as a function of time with temporal resolution well below 100 fs. This capability is based on the pump-probe technique where one optical pulse triggers a reaction and a second delayed optical pulse probes the changes that ensue. To illustrate this capability, the dynamics of ligand motion within a protein are presented. Moving beyond casual observation of a reaction to active control of its outcome requires additional experimental and theoretical effort. To illustrate the concept of control, the effect of optical pulse duration on the vibrational dynamics of a tri-atomic molecule are discussed. The experimental and theoretical resources currently available are poised to make the dream of reaction control a reality for certain molecular systems.
Assuntos
Mioglobina/química , Animais , Monóxido de Carbono/química , Cinética , Ligantes , Fotólise , Espectrofotometria InfravermelhoRESUMO
The topic of femtochemistry is surveyed from both theoretical and experimental points of view. A time-dependent wave packet description of the photodissociation of the O---C---S molecule reveals vibrational motion in the transition-state region and suggests targets for direct experimental observation. Theoretical approaches for treating femtosecond chemical phenomena in condensed phases are featured along with prospects for laser-controlled chemical reactions by using tailored ultrashort chirped pulses. An experimental study of the photoisomerization of retinal in the protein bacteriorhodopsin is discussed with an aim to gain insight into the potential energy surfaces on which this remarkably efficient and selective reactions proceeds. Finally, a prospective view of new frontiers in femtochemistry is given.
Assuntos
Bacteriorodopsinas/química , Microquímica/tendências , Bacteriorodopsinas/metabolismo , Cristalografia por Raios X , Halobacterium salinarum/metabolismo , Mioglobina/química , Mioglobina/metabolismo , Retinaldeído/química , Retinaldeído/metabolismoRESUMO
The vibrational energy relaxation of carbon monoxide in the heme pocket of sperm whale myoglobin was studied by using molecular dynamics simulation and normal mode analysis methods. Molecular dynamics trajectories of solvated myoglobin were run at 300 K for both the delta- and epsilon-tautomers of the distal His-64. Vibrational population relaxation times of 335 +/- 115 ps for the delta-tautomer and 640 +/- 185 ps for the epsilon-tautomer were estimated by using the Landau-Teller model. Normal mode analysis was used to identify those protein residues that act as the primary "doorway" modes in the vibrational relaxation of the oscillator. Although the CO relaxation rates in both the epsilon- and delta-tautomers are similar in magnitude, the simulations predict that the vibrational relaxation of the CO is faster in the delta-tautomer with the distal His playing an important role in the energy relaxation mechanism. Time-resolved mid-IR absorbance measurements were performed on photolyzed carbonmonoxy hemoglobin (Hb(13)CO). From these measurements, a T(1) time of 600 +/- 150 ps was determined. The simulation and experimental estimates are compared and discussed.