RESUMO
Protein X-ray structures are determined with ionizing radiation that damages the protein at high X-ray doses. As a result, diffraction patterns deteriorate with the increased absorbed dose. Several strategies such as sample freezing or scavenging of X-ray-generated free radicals are currently employed to minimize this damage. However, little is known about how the absorbed X-ray dose affects time-resolved Laue data collected at physiological temperatures where the protein is fully functional in the crystal, and how the kinetic analysis of such data depends on the absorbed dose. Here, direct evidence for the impact of radiation damage on the function of a protein is presented using time-resolved macromolecular crystallography. The effect of radiation damage on the kinetic analysis of time-resolved X-ray data is also explored.
Assuntos
Proteínas de Bactérias/química , Proteínas de Bactérias/efeitos da radiação , Fotorreceptores Microbianos/química , Fotorreceptores Microbianos/efeitos da radiação , Proteínas/efeitos da radiação , Cristalografia por Raios X , Cinética , Substâncias Macromoleculares/química , Conceitos Matemáticos , Proteínas/química , Temperatura , Raios XRESUMO
Serial x-ray crystallography can uncover binding events, and subsequent chemical conversions occurring during enzymatic reaction. Here, we reveal the structure, binding and cleavage of moxalactam antibiotic bound to L1 metallo-ß-lactamase (MBL) from Stenotrophomonas maltophilia. Using time-resolved serial synchrotron crystallography, we show the time course of ß-lactam hydrolysis and determine ten snapshots (20, 40, 60, 80, 100, 150, 300, 500, 2000 and 4000 ms) at 2.20 Å resolution. The reaction is initiated by laser pulse releasing Zn2+ ions from a UV-labile photocage. Two metal ions bind to the active site, followed by binding of moxalactam and the intact ß-lactam ring is observed for 100 ms after photolysis. Cleavage of ß-lactam is detected at 150 ms and the ligand is significantly displaced. The reaction product adjusts its conformation reaching steady state at 2000 ms corresponding to the relaxed state of the enzyme. Only small changes are observed in the positions of Zn2+ ions and the active site residues. Mechanistic details captured here can be generalized to other MBLs.
Assuntos
Moxalactam , beta-Lactamas , beta-Lactamases , Cristalografia por Raios XRESUMO
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 biological activity of macromolecules is accompanied by rapid structural changes. The photosensitivity of the carbon monoxide complex of myoglobin was used at the European Synchrotron Radiation Facility to obtain pulsed, Laue x-ray diffraction data with nanosecond time resolution during the process of heme and protein relaxation after carbon monoxide photodissociation and during rebinding. These time-resolved experiments reveal the structures of myoglobin photoproducts, provide a structural foundation to spectroscopic results and molecular dynamics calculations, and demonstrate that time-resolved macromolecular crystallography can elucidate the structural bases of biochemical mechanisms on the nanosecond time scale.
Assuntos
Cristalografia por Raios X/métodos , Mioglobina/química , Monóxido de Carbono/química , Monóxido de Carbono/metabolismo , Simulação por Computador , Análise de Fourier , Globinas/química , Heme/química , Histidina/química , Ferro/química , Ligantes , Mioglobina/metabolismo , Fotólise , Temperatura , Fatores de TempoRESUMO
The blue-light photoreceptor photoactive yellow protein (PYP) undergoes a self-contained light cycle. The atomic structure of the bleached signaling intermediate in the light cycle of PYP was determined by millisecond time-resolved, multiwavelength Laue crystallography and simultaneous optical spectroscopy. Light-induced trans-to-cis isomerization of the 4-hydroxycinnamyl chromophore and coupled protein rearrangements produce a new set of active-site hydrogen bonds. An arginine gateway opens, allowing solvent exposure and protonation of the chromophore's phenolic oxygen. Resulting changes in shape, hydrogen bonding, and electrostatic potential at the protein surface form a likely basis for signal transduction. The structural results suggest a general framework for the interpretation of protein photocycles.
Assuntos
Proteínas de Bactérias/química , Fotorreceptores Microbianos , Conformação Proteica , Proteínas de Bactérias/fisiologia , Sítios de Ligação , Chromatiaceae , Cristalografia por Raios X , Eletroquímica , Ligação de Hidrogênio , Isomerismo , Luz , Modelos Moleculares , Transdução de Sinais , Análise EspectralRESUMO
Photoactive yellow protein (PYP) is a member of the xanthopsin family of eubacterial blue-light photoreceptors. On absorption of light, PYP enters a photocycle that ultimately transduces the energy contained in a light signal into an altered biological response. Nanosecond time-resolved x-ray crystallography was used to determine the structure of the short-lived, red-shifted, intermediate state denoted [pR], which develops within 1 nanosecond after photoelectronic excitation of the chromophore of PYP by absorption of light. The resulting structural model demonstrates that the [pR] state possesses the cis conformation of the 4-hydroxyl cinnamic thioester chromophore, and that the process of trans to cis isomerization is accompanied by the specific formation of new hydrogen bonds that replace those broken upon excitation of the chromophore. Regions of flexibility that compose the chromophore-binding pocket serve to lower the activation energy barrier between the dark state, denoted pG, and [pR], and help initiate entrance into the photocycle. Direct structural evidence is provided for the initial processes of transduction of light energy, which ultimately translate into a physiological signal.
Assuntos
Proteínas de Bactérias/química , Luz , Fotorreceptores Microbianos , Conformação Proteica , Proteínas de Bactérias/metabolismo , Chromatiaceae/química , Cristalografia por Raios X , Metabolismo Energético , Análise de Fourier , Ligação de Hidrogênio , Isomerismo , Cinética , Modelos Moleculares , Transdução de SinaisRESUMO
Serial X-ray crystallography allows macromolecular structure determination at both X-ray free electron lasers (XFELs) and, more recently, synchrotron sources. The time resolution for serial synchrotron crystallography experiments has been limited to millisecond timescales with monochromatic beams. The polychromatic, "pink", beam provides a more than two orders of magnitude increased photon flux and hence allows accessing much shorter timescales in diffraction experiments at synchrotron sources. Here we report the structure determination of two different protein samples by merging pink-beam diffraction patterns from many crystals, each collected with a single 100 ps X-ray pulse exposure per crystal using a setup optimized for very low scattering background. In contrast to experiments with monochromatic radiation, data from only 50 crystals were required to obtain complete datasets. The high quality of the diffraction data highlights the potential of this method for studying irreversible reactions at sub-microsecond timescales using high-brightness X-ray facilities.
Assuntos
Cristalografia por Raios X/métodos , Cristalografia por Raios X/instrumentação , Cristalografia por Raios X/estatística & dados numéricos , Bases de Dados de Compostos Químicos/estatística & dados numéricos , Endopeptidase K/química , Desenho de Equipamento , Modelos Moleculares , Ficocianina/química , Conformação Proteica , Eletricidade Estática , Síncrotrons , Difração de Raios XRESUMO
We present the results of an extensive investigation of the optical line shapes of deoxymyoglobin (Mb), the ligand-bound form (MbCO), and the low-temperature photoproduct (Mb*). The thermal properties and the pH dependence of the Soret band and the near infrared band III (approximately 760 nm) are analyzed, taking into account the underlying vibrational properties of the absorption bands. The strong temperature dependence associated with the Soret band of MbCO and band III of Mb indicates significant coupling to low-frequency modes that may not be directly observed in the resonance Raman spectra. On the basis of analogous line-shape studies in a variety of heme systems, we assign the low-frequency coupling in MbCO to torsional motions of the CO molecule. The low-frequency mode coupled to band III (approximately 70 cm-1) is found to lie quite close to the value for the heme-doming motion (approximately 50 cm-1) calculated by using the kinetically determined value of the force constant (17 N/m). Significant inhomogeneous broadening in the Soret region of Mb and Mb* is found to be due to a "nonkinetic" coordinate that we associate with the orientation of the proximal histidine. A "kinetic" coordinate, associated with the equilibrium displacement of the iron atom from the porphyrin plane (a) is found to contribute to the inhomogeneous broadening of both the Soret band and band III. The relaxation of the heme as the system evolves from from Mb* to Mb is followed optically as a function of temperature, and a sharp transition temperature is found at 185 K. The blue shifts of the Soret band and band III as Mb* evolves to Mb are found to be nearly identical (delta v*ABS approximately 140 cm-1) and attributed to changes in the mean value of a between Mb* (a*0) and Mb (a0 = 0.45 A). A simple quadratic model for the coordinate coupling that simultaneously accounts for the observed shift, delta v*ABS, the low-temperature kinetics and the kinetic hole burning predicts a*0 = 0.2 +/- 0.05 A and EA = 16 +/- 2 kJ/mol for the room temperature Arrhenius barrier height at the heme. A simple quantitative method for the analysis of kinetic hole-burning experiments is also developed and applied to recent studies involving quaternary and subunit-specific hemoglobin structures.
Assuntos
Mioglobina/análogos & derivados , Mioglobina/química , Monóxido de Carbono/metabolismo , Histidina , Concentração de Íons de Hidrogênio , Ferro , Cinética , Substâncias Macromoleculares , Matemática , Mioglobina/metabolismo , Oxigênio , Conformação Proteica , Espectrofotometria , Análise Espectral Raman , TemperaturaRESUMO
Myoglobin's reversible binding of oxygen is a model for studies of protein control of ligand binding and discrimination. Protein relaxation and geminate ligand rebinding subsequent to ligand photodissociation have been studied extensively by a variety of techniques. The ps to ns time scales for these processes are still much shorter than the ms time resolution of X-ray diffraction experiments, but it may be possible to trap these intermediates at low temperatures. We report here an X-ray diffraction investigation of structural changes induced by photolysis of carbonmonoxy myoglobin crystals at 40 K. Our results provide a structural basis for the interpretation of ambient and low temperature spectroscopic observations and molecular dynamics simulations of the ligand photodissociation and binding processes in haem proteins.
Assuntos
Mioglobina/química , Animais , Temperatura Baixa , Gráficos por Computador , Cristalografia por Raios X , Análise de Fourier , Mioglobina/metabolismo , Oxigênio/metabolismo , Fotólise , Ligação Proteica , Conformação Proteica , BaleiasRESUMO
We present evidence from resonance Raman and absorption measurements that the extended exposure of MbCO to CW laser light at low temperatures alters the CO rebinding kinetics and leads to a significantly increased population of very long lived states of photolyzed MbCO. This optical "pumping" process is observed for samples frozen in both aqueous buffer and glycerol/buffer and exhibits power law behavior with a very weak temperature dependence. A comparison of the nonexponential rebinding kinetics of CO molecules from the pumped states with the rebinding observed in flash photolysis experiments suggests that the pumped states are distinct geminate states, not observed in flash photolysis experiments. Thus, a four-state model, with two geminate states, is implicated for MbCO. Pumped states may represent "separated geminate pair" states with the CO molecule still in the heme pocket or possibly trapped within a cavity on its way through the protein matrix, consistent with molecular dynamics simulations. The possibility of significant deoxyheme relaxation from a less domed to a more domed configuration, as a result of the multiple photolysis events associated with the pumping process, is also explored. However, the small changes observed in the Soret band line shape and position subsequent to pumping at T less than 180 K tend to rule out this explanation for the pumping process. Since the yield for creating a pumped state is small (e.g., less than 10(-7) for T greater than 100 K), pumping can be observed only after extended illumination and is absent in flash photolysis measurements, even after multiple flashes. At higher temperatures (T greater than 180 K), the escape of the CO molecule to the solvent is observed. Our data are consistent with a "phase transition" of the protein that is coupled to the surrounding matrix. The protein fluctuations are quenched below approximately 185 K for a solvent composed of 70% glycerol and below approximately 260 K for aqueous buffer. We also present the first large amplitude measurements of CO rebinding from the protein exterior, observed below 200 K after freezing the sample under laser illumination.
Assuntos
Mioglobina/metabolismo , Animais , Heme/metabolismo , Cinética , Lasers , Matemática , Mioglobina/química , Mioglobina/efeitos da radiação , Fotólise , Ligação Proteica , Solventes , Espectrofotometria , Análise Espectral Raman , Termodinâmica , Fatores de Tempo , BaleiasRESUMO
Migration of the CO ligand following photolysis of carbonmonoxy myoglobin (MbCO) in single crystals has been investigated by time-resolved X-ray diffraction at 40K. After short illumination by weak visible light at a photolysis rate of approximately 1 s-1, the photodissociated CO molecule is found about 1 A from its bound location. After continuous illumination over several hours, the CO molecule migrates to a more distant site in the distal pocket, about 2.5 A from its bound location. Migration of the ligand under continuous illumination accounts for different locations of the photodissociated CO molecule previously reported in three cryocrystallographic studies [Teng, T.-Y., et al. (1994) Nat. Struct. Biol. 1, 701-705; Schlichting, I., et al. (1994) Nature 371, 808-812; Hartmann, H., et al. (1996) Proc.Natl.Acad. Sci. U.S.A. 93, 7013-7016]. Due to the different photolysis protocols employed in these studies, each reveals a different part of the trajectory of the photodissociated CO molecule. When the different experimental parts of the trajectory at 40 K are pieced together and combined with our nanosecond time-resolved studies at room temperature [Srajer, V., et al. (1996) Science 274, 1726-1729], excellent agreement is obtained with recent theoretical predictions of the CO probability distribution in the ligand pocket [Vitkup, D., et al. (1997) Nat. Struct. Biol. 4, 202-208]. The heme relaxation that accompanies ligand photolysis is incomplete, about 30% of that associated with the conversion of MbCO to deoxy-Mb at room temperature, and independent of the duration of illumination. Other tertiary structural changes in the globin are also greatly diminished. The globin structure is therefore very rigid at cryogenic temperatures, and structural relaxation is greatly hindered, consistent with numerous spectroscopic measurements.
Assuntos
Monóxido de Carbono/química , Congelamento , Mioglobina/química , Fotólise , Cristalografia por Raios X/métodos , Heme/química , Temperatura Alta , Lasers , Ligantes , Estrutura Terciária de ProteínaRESUMO
Wavelength normalization is an essential part of processing of Laue X-ray diffraction data and is critically important for deriving accurate structure-factor amplitudes. The results of wavelength normalization for Laue data obtained in nanosecond time-resolved experiments at the ID09 beamline at the European Synchrotron Radiation Facility, Grenoble, France, are presented. Several wiggler and undulator insertion devices with complex spectra were used. The results show that even in the most challenging cases, such as wiggler/undulator tandems or single-line undulators, accurate wavelength normalization does not require unusually redundant Laue data and can be accomplished using typical Laue data sets. Single-line undulator spectra derived from Laue data compare well with the measured incident X-ray spectra. Successful wavelength normalization of the undulator data was also confirmed by the observed signal in nanosecond time-resolved experiments. Single-line undulators, which are attractive for time-resolved experiments due to their high peak intensity and low polychromatic background, are compared with wigglers, based on data obtained on the same crystal.
RESUMO
Laue diffraction patterns with an exposure time of ca 60 ps have been acquired at the European Synchrotron Radiation Facility (ESRF) on protein crystals by using the single-bunch mode of the storage ring. A 10 ns laser pulse initiating photodissociation was synchronized with the X-ray pulse. The potential for a quantitative detection of conformational changes in proteins on the nanosecond timescale with this technique is demonstrated using the example of carbonmonoxymyoglobin, from simulations and real data. The instrumental aspects of the experiment (highly intense X-ray beam, fast shutter system, Laue camera, detector, laser apparatus and synchronization technique) are emphasized.
RESUMO
The photocycle of the bacterial blue-light photoreceptor, photoactive yellow protein, was stimulated by illumination of single crystals by a 7 ns laser pulse. The molecular events were recorded at high resolution by time-resolved X-ray Laue diffraction as they evolved in real time, from 1 ns to seconds after the laser pulse. The complex structural changes during the photocycle at ambient temperature are displayed in a movie of difference electron density maps relative to the dark state. The step critical to entry into the photocycle is identified as flipping of the carbonyl group of the 4-hydroxycinnamic acid chromophore into an adjacent, hydrophobic environment rather than the concomitant isomerization about the double bond of the chromophore tail. The structural perturbation generated at the chromophore propagates throughout the entire protein as a light-induced "protein quake" with its "epicenter" at the carbonyl moiety of the chromophore.
Assuntos
Proteínas de Bactérias/química , Cristalografia por Raios X/métodos , Halorhodospira halophila/química , Modelos Moleculares , Fotoperíodo , Fotorreceptores Microbianos/química , Simulação por Computador , Cristalografia por Raios X/instrumentação , Ligação de Hidrogênio , Oxigênio/química , Soluções , Termodinâmica , Fatores de TempoRESUMO
A time-resolved Laue X-ray diffraction technique has been used to explore protein relaxation and ligand migration at room temperature following photolysis of a single crystal of carbon monoxymyoglobin. The CO ligand is photodissociated by a 7.5 ns laser pulse, and the subsequent structural changes are probed by 150 ps or 1 micros X-ray pulses at 14 laser/X-ray delay times, ranging from 1 ns to 1.9 ms. Very fast heme and protein relaxation involving the E and F helices is evident from the data at a 1 ns time delay. The photodissociated CO molecules are detected at two locations: at a distal pocket docking site and at the Xe 1 binding site in the proximal pocket. The population by CO of the primary, distal site peaks at a 1 ns time delay and decays to half the peak value in 70 ns. The secondary, proximal docking site reaches its highest occupancy of 20% at approximately 100 ns and has a half-life of approximately 10 micros. At approximately 100 ns, all CO molecules are accounted for within the protein: in one of these two docking sites or bound to the heme. Thereafter, the CO molecules migrate to the solvent from which they rebind to deoxymyoglobin in a bimolecular process with a second-order rate coefficient of 4.5 x 10(5) M(-1) s(-1). Our results also demonstrate that structural changes as small as 0.2 A and populations of CO docking sites of 10% can be detected by time-resolved X-ray diffraction.