RESUMEN
Angiotensin II type 1 receptor (AT(1)R) is a G protein-coupled receptor that serves as a primary regulator for blood pressure maintenance. Although several anti-hypertensive drugs have been developed as AT(1)R blockers (ARBs), the structural basis for AT(1)R ligand-binding and regulation has remained elusive, mostly due to the difficulties of growing high-quality crystals for structure determination using synchrotron radiation. By applying the recently developed method of serial femtosecond crystallography at an X-ray free-electron laser, we successfully determined the room-temperature crystal structure of the human AT(1)R in complex with its selective antagonist ZD7155 at 2.9-Å resolution. The AT(1)R-ZD7155 complex structure revealed key structural features of AT(1)R and critical interactions for ZD7155 binding. Docking simulations of the clinically used ARBs into the AT(1)R structure further elucidated both the common and distinct binding modes for these anti-hypertensive drugs. Our results thereby provide fundamental insights into AT(1)R structure-function relationship and structure-based drug design.
Asunto(s)
Bloqueadores del Receptor Tipo 1 de Angiotensina II/farmacología , Receptor de Angiotensina Tipo 1/química , Secuencia de Aminoácidos , Bloqueadores del Receptor Tipo 1 de Angiotensina II/química , Cristalografía por Rayos X , Humanos , Datos de Secuencia Molecular , Mutagénesis , Naftiridinas/química , Naftiridinas/farmacología , Receptor de Angiotensina Tipo 1/genética , Receptor de Angiotensina Tipo 1/metabolismo , Alineación de SecuenciaRESUMEN
The human MT1 and MT2 melatonin receptors1,2 are G-protein-coupled receptors (GPCRs) that help to regulate circadian rhythm and sleep patterns3. Drug development efforts have targeted both receptors for the treatment of insomnia, circadian rhythm and mood disorders, and cancer3, and MT2 has also been implicated in type 2 diabetes4,5. Here we report X-ray free electron laser (XFEL) structures of the human MT2 receptor in complex with the agonists 2-phenylmelatonin (2-PMT) and ramelteon6 at resolutions of 2.8 Å and 3.3 Å, respectively, along with two structures of function-related mutants: H2085.46A (superscripts represent the Ballesteros-Weinstein residue numbering nomenclature7) and N862.50D, obtained in complex with 2-PMT. Comparison of the structures of MT2 with a published structure8 of MT1 reveals that, despite conservation of the orthosteric ligand-binding site residues, there are notable conformational variations as well as differences in [3H]melatonin dissociation kinetics that provide insights into the selectivity between melatonin receptor subtypes. A membrane-buried lateral ligand entry channel is observed in both MT1 and MT2, but in addition the MT2 structures reveal a narrow opening towards the solvent in the extracellular part of the receptor. We provide functional and kinetic data that support a prominent role for intramembrane ligand entry in both receptors, and suggest that there might also be an extracellular entry path in MT2. Our findings contribute to a molecular understanding of melatonin receptor subtype selectivity and ligand access modes, which are essential for the design of highly selective melatonin tool compounds and therapeutic agents.
Asunto(s)
Electrones , Rayos Láser , Modelos Moleculares , Receptor de Melatonina MT2/química , Receptor de Melatonina MT2/metabolismo , Cristalización , Diabetes Mellitus Tipo 2/genética , Humanos , Indenos/química , Indenos/metabolismo , Ligandos , Melatonina/análogos & derivados , Melatonina/química , Melatonina/metabolismo , Simulación del Acoplamiento Molecular , Simulación de Dinámica Molecular , Mutación , Receptor de Melatonina MT1/química , Receptor de Melatonina MT1/metabolismo , Receptor de Melatonina MT2/genética , Relación Estructura-Actividad , Especificidad por SustratoRESUMEN
Melatonin (N-acetyl-5-methoxytryptamine) is a neurohormone that maintains circadian rhythms1 by synchronization to environmental cues and is involved in diverse physiological processes2 such as the regulation of blood pressure and core body temperature, oncogenesis, and immune function3. Melatonin is formed in the pineal gland in a light-regulated manner4 by enzymatic conversion from 5-hydroxytryptamine (5-HT or serotonin), and modulates sleep and wakefulness5 by activating two high-affinity G-protein-coupled receptors, type 1A (MT1) and type 1B (MT2)3,6. Shift work, travel, and ubiquitous artificial lighting can disrupt natural circadian rhythms; as a result, sleep disorders affect a substantial population in modern society and pose a considerable economic burden7. Over-the-counter melatonin is widely used to alleviate jet lag and as a safer alternative to benzodiazepines and other sleeping aids8,9, and is one of the most popular supplements in the United States10. Here, we present high-resolution room-temperature X-ray free electron laser (XFEL) structures of MT1 in complex with four agonists: the insomnia drug ramelteon11, two melatonin analogues, and the mixed melatonin-serotonin antidepressant agomelatine12,13. The structure of MT2 is described in an accompanying paper14. Although the MT1 and 5-HT receptors have similar endogenous ligands, and agomelatine acts on both receptors, the receptors differ markedly in the structure and composition of their ligand pockets; in MT1, access to the ligand pocket is tightly sealed from solvent by extracellular loop 2, leaving only a narrow channel between transmembrane helices IV and V that connects it to the lipid bilayer. The binding site is extremely compact, and ligands interact with MT1 mainly by strong aromatic stacking with Phe179 and auxiliary hydrogen bonds with Asn162 and Gln181. Our structures provide an unexpected example of atypical ligand entry for a non-lipid receptor, lay the molecular foundation of ligand recognition by melatonin receptors, and will facilitate the design of future tool compounds and therapeutic agents, while their comparison to 5-HT receptors yields insights into the evolution and polypharmacology of G-protein-coupled receptors.
Asunto(s)
Electrones , Rayos Láser , Modelos Moleculares , Receptor de Melatonina MT1/química , Receptor de Melatonina MT1/metabolismo , Acetamidas/química , Acetamidas/metabolismo , Secuencia de Aminoácidos , Antidepresivos/química , Antidepresivos/metabolismo , Cristalización , Humanos , Indenos/química , Indenos/metabolismo , Ligandos , Melatonina/análogos & derivados , Melatonina/química , Simulación del Acoplamiento Molecular , Mutación , Receptor de Melatonina MT1/agonistas , Receptor de Melatonina MT1/genética , Receptor de Serotonina 5-HT2C/química , Relación Estructura-Actividad , Especificidad por SustratoRESUMEN
Change history: In this Letter, the rotation signs around 90°, 135° and 15° were missing and in the HTML, Extended Data Tables 2 and 3 were the wrong tables; these errors have been corrected online.
RESUMEN
Prostaglandin D2 (PGD2) signals through the G protein-coupled receptor (GPCR) CRTH2 to mediate various inflammatory responses. CRTH2 is the only member of the prostanoid receptor family that is phylogenetically distant from others, implying a nonconserved mechanism of lipid action on CRTH2. Here, we report a crystal structure of human CRTH2 bound to a PGD2 derivative, 15R-methyl-PGD2 (15mPGD2), by serial femtosecond crystallography. The structure revealed a "polar group in"-binding mode of 15mPGD2 contrasting the "polar group out"-binding mode of PGE2 in its receptor EP3. Structural comparison analysis suggested that these two lipid-binding modes, associated with distinct charge distributions of ligand-binding pockets, may apply to other lipid GPCRs. Molecular dynamics simulations together with mutagenesis studies also identified charged residues at the ligand entry port that function to capture lipid ligands of CRTH2 from the lipid bilayer. Together, our studies suggest critical roles of charge environment in lipid recognition by GPCRs.
Asunto(s)
Receptores Inmunológicos/química , Receptores Inmunológicos/metabolismo , Receptores de Prostaglandina/química , Receptores de Prostaglandina/metabolismo , Cristalografía por Rayos X/métodos , Humanos , Metabolismo de los Lípidos , Simulación de Dinámica Molecular , Mutación , Prostaglandina D2/química , Prostaglandina D2/metabolismo , Conformación Proteica , Receptores Inmunológicos/genética , Receptores de Prostaglandina/genéticaRESUMEN
Chloride ion-pumping rhodopsin (ClR) in some marine bacteria utilizes light energy to actively transport Cl- into cells. How the ClR initiates the transport is elusive. Here, we show the dynamics of ion transport observed with time-resolved serial femtosecond (fs) crystallography using the Linac Coherent Light Source. X-ray pulses captured structural changes in ClR upon flash illumination with a 550 nm fs-pumping laser. High-resolution structures for five time points (dark to 100 ps after flashing) reveal complex and coordinated dynamics comprising retinal isomerization, water molecule rearrangement, and conformational changes of various residues. Combining data from time-resolved spectroscopy experiments and molecular dynamics simulations, this study reveals that the chloride ion close to the Schiff base undergoes a dissociation-diffusion process upon light-triggered retinal isomerization.
Asunto(s)
Canales de Cloruro/metabolismo , Cloruros/metabolismo , Rodopsinas Microbianas/metabolismo , Cationes Monovalentes/metabolismo , Canales de Cloruro/aislamiento & purificación , Canales de Cloruro/efectos de la radiación , Canales de Cloruro/ultraestructura , Cristalografía/métodos , Radiación Electromagnética , Rayos Láser , Simulación de Dinámica Molecular , Nocardioides , Conformación Proteica en Hélice alfa/efectos de la radiación , Estructura Terciaria de Proteína/efectos de la radiación , Proteínas Recombinantes/aislamiento & purificación , Proteínas Recombinantes/metabolismo , Proteínas Recombinantes/efectos de la radiación , Proteínas Recombinantes/ultraestructura , Retinaldehído/metabolismo , Retinaldehído/efectos de la radiación , Rodopsinas Microbianas/aislamiento & purificación , Rodopsinas Microbianas/efectos de la radiación , Rodopsinas Microbianas/ultraestructura , Agua/metabolismoRESUMEN
The angiotensin II receptors AT1R and AT2R serve as key components of the renin-angiotensin-aldosterone system. AT1R has a central role in the regulation of blood pressure, but the function of AT2R is unclear and it has a variety of reported effects. To identify the mechanisms that underlie the differences in function and ligand selectivity between these receptors, here we report crystal structures of human AT2R bound to an AT2R-selective ligand and to an AT1R/AT2R dual ligand, capturing the receptor in an active-like conformation. Unexpectedly, helix VIII was found in a non-canonical position, stabilizing the active-like state, but at the same time preventing the recruitment of G proteins or ß-arrestins, in agreement with the lack of signalling responses in standard cellular assays. Structure-activity relationship, docking and mutagenesis studies revealed the crucial interactions for ligand binding and selectivity. Our results thus provide insights into the structural basis of the distinct functions of the angiotensin receptors, and may guide the design of new selective ligands.
Asunto(s)
Modelos Moleculares , Receptor de Angiotensina Tipo 2/química , Receptor de Angiotensina Tipo 2/metabolismo , Bloqueadores del Receptor Tipo 2 de Angiotensina II/química , Bloqueadores del Receptor Tipo 2 de Angiotensina II/metabolismo , Sitios de Unión/genética , Cristalografía por Rayos X , Diseño de Fármacos , Proteínas de Unión al GTP Heterotriméricas/química , Proteínas de Unión al GTP Heterotriméricas/metabolismo , Humanos , Ligandos , Simulación del Acoplamiento Molecular , Mutación , Unión Proteica , Conformación Proteica , Receptor de Angiotensina Tipo 1/química , Receptor de Angiotensina Tipo 1/metabolismo , Receptor de Angiotensina Tipo 2/agonistas , Receptor de Angiotensina Tipo 2/genética , Transducción de Señal , Relación Estructura-Actividad , Especificidad por Sustrato/genética , beta-Arrestinas/metabolismoRESUMEN
The human glucagon receptor, GCGR, belongs to the class B G-protein-coupled receptor family and plays a key role in glucose homeostasis and the pathophysiology of type 2 diabetes. Here we report the 3.0 Å crystal structure of full-length GCGR containing both the extracellular domain and transmembrane domain in an inactive conformation. The two domains are connected by a 12-residue segment termed the stalk, which adopts a ß-strand conformation, instead of forming an α-helix as observed in the previously solved structure of the GCGR transmembrane domain. The first extracellular loop exhibits a ß-hairpin conformation and interacts with the stalk to form a compact ß-sheet structure. Hydrogen-deuterium exchange, disulfide crosslinking and molecular dynamics studies suggest that the stalk and the first extracellular loop have critical roles in modulating peptide ligand binding and receptor activation. These insights into the full-length GCGR structure deepen our understanding of the signalling mechanisms of class B G-protein-coupled receptors.
Asunto(s)
Receptores de Glucagón/química , Receptores de Glucagón/clasificación , Sitio Alostérico/efectos de los fármacos , Benzamidas/química , Benzamidas/metabolismo , Benzamidas/farmacología , Membrana Celular/metabolismo , Reactivos de Enlaces Cruzados/química , Cristalografía por Rayos X , Medición de Intercambio de Deuterio , Disulfuros/química , Humanos , Ligandos , Modelos Moleculares , Simulación de Dinámica Molecular , Compuestos de Fenilurea/química , Compuestos de Fenilurea/metabolismo , Compuestos de Fenilurea/farmacología , Dominios Proteicos , Estabilidad Proteica , Receptores de Glucagón/agonistas , Receptores de Glucagón/metabolismoRESUMEN
A revised version of Table 2 of Martin-Garcia et al. [J. Synchrotron Rad. (2022). 29, 896-907] is provided.
RESUMEN
The increase in successful adaptations of serial crystallography at synchrotron radiation sources continues. To date, the number of serial synchrotron crystallography (SSX) experiments has grown exponentially, with over 40 experiments reported so far. In this work, we report the first SSX experiments with viscous jets conducted at ALBA beamline BL13-XALOC. Small crystals (15-30â µm) of five soluble proteins (lysozyme, proteinase K, phycocyanin, insulin and α-spectrin-SH3 domain) were suspended in lipidic cubic phase (LCP) and delivered to the X-ray beam with a high-viscosity injector developed at Arizona State University. Complete data sets were collected from all proteins and their high-resolution structures determined. The high quality of the diffraction data collected from all five samples, and the lack of specific radiation damage in the structures obtained in this study, confirm that the current capabilities at the beamline enables atomic resolution determination of protein structures from microcrystals as small as 15â µm using viscous jets at room temperature. Thus, BL13-XALOC can provide a feasible alternative to X-ray free-electron lasers when determining snapshots of macromolecular structures.
Asunto(s)
Rayos Láser , Sincrotrones , Cristalografía por Rayos X , Humanos , Sustancias Macromoleculares , Proteínas , ViscosidadRESUMEN
The three-dimensional structures of macromolecules and their complexes are mainly elucidated by X-ray protein crystallography. A major limitation of this method is access to high-quality crystals, which is necessary to ensure X-ray diffraction extends to sufficiently large scattering angles and hence yields information of sufficiently high resolution with which to solve the crystal structure. The observation that crystals with reduced unit-cell volumes and tighter macromolecular packing often produce higher-resolution Bragg peaks suggests that crystallographic resolution for some macromolecules may be limited not by their heterogeneity, but by a deviation of strict positional ordering of the crystalline lattice. Such displacements of molecules from the ideal lattice give rise to a continuous diffraction pattern that is equal to the incoherent sum of diffraction from rigid individual molecular complexes aligned along several discrete crystallographic orientations and that, consequently, contains more information than Bragg peaks alone. Although such continuous diffraction patterns have long been observed--and are of interest as a source of information about the dynamics of proteins--they have not been used for structure determination. Here we show for crystals of the integral membrane protein complex photosystem II that lattice disorder increases the information content and the resolution of the diffraction pattern well beyond the 4.5-ångström limit of measurable Bragg peaks, which allows us to phase the pattern directly. Using the molecular envelope conventionally determined at 4.5 ångströms as a constraint, we obtain a static image of the photosystem II dimer at a resolution of 3.5 ångströms. This result shows that continuous diffraction can be used to overcome what have long been supposed to be the resolution limits of macromolecular crystallography, using a method that exploits commonly encountered imperfect crystals and enables model-free phasing.
Asunto(s)
Cristalografía por Rayos X/métodos , Complejo de Proteína del Fotosistema II/química , Cristalización , Modelos MolecularesRESUMEN
Misoprostol is a life-saving drug in many developing countries for women at risk of post-partum hemorrhaging owing to its affordability, stability, ease of administration and clinical efficacy. However, misoprostol lacks receptor and tissue selectivities, and thus its use is accompanied by a number of serious side effects. The development of pharmacological agents combining the advantages of misoprostol with improved selectivity is hindered by the absence of atomic details of misoprostol action in labor induction. Here, we present the 2.5 Å resolution crystal structure of misoprostol free-acid form bound to the myometrium labor-inducing prostaglandin E2 receptor 3 (EP3). The active state structure reveals a completely enclosed binding pocket containing a structured water molecule that coordinates misoprostol's ring structure. Modeling of selective agonists in the EP3 structure reveals rationales for selectivity. These findings will provide the basis for the next generation of uterotonic drugs that will be suitable for administration in low resource settings.
Asunto(s)
Misoprostol/química , Subtipo EP3 de Receptores de Prostaglandina E/química , Subtipo EP3 de Receptores de Prostaglandina E/metabolismo , Sitios de Unión , Cristalografía por Rayos X , Dinoprostona/análogos & derivados , Dinoprostona/química , Dinoprostona/metabolismo , Humanos , Misoprostol/metabolismo , Simulación del Acoplamiento Molecular , Mutagénesis Sitio-Dirigida , Conformación Proteica , Subtipo EP3 de Receptores de Prostaglandina E/agonistas , Subtipo EP3 de Receptores de Prostaglandina E/genética , Transducción de Señal , Agua/químicaRESUMEN
In the version of this article originally published, the present address for Petr Popov was incorrectly listed as 'Koltech Institute of Science & Technology, Moscow, Russia'. The correct present address is 'Skolkovo Institute of Science and Technology, Moscow, Russia'. The error has been corrected in the HTML and PDF versions of the paper.
RESUMEN
Non-cryogenic protein structures determined at ambient temperature may disclose significant information about protein activity. Chloride-pumping rhodopsin (ClR) exhibits a trend to hyperactivity induced by a change in the photoreaction rate because of a gradual decrease in temperature. Here, to track the structural changes that explain the differences in CIR activity resulting from these temperature changes, we used serial femtosecond crystallography (SFX) with an X-ray free electron laser (XFEL) to determine the non-cryogenic structure of ClR at a resolution of 1.85 Å, and compared this structure with a cryogenic ClR structure obtained with synchrotron X-ray crystallography. The XFEL-derived ClR structure revealed that the all-trans retinal (ATR) region and positions of two coordinated chloride ions slightly differed from those of the synchrotron-derived structure. Moreover, the XFEL structure enabled identification of one additional water molecule forming a hydrogen bond network with a chloride ion. Analysis of the channel cavity and a difference distance matrix plot (DDMP) clearly revealed additional structural differences. B-factor information obtained from the non-cryogenic structure supported a motility change on the residual main and side chains as well as of chloride and water molecules because of temperature effects. Our results indicate that non-cryogenic structures and time-resolved XFEL experiments could contribute to a better understanding of the chloride-pumping mechanism of ClR and other ion pumps.
Asunto(s)
Actinomycetales/química , Canales de Cloruro/química , Rodopsinas Microbianas/química , Cristalografía por Rayos X , Dominios ProteicosRESUMEN
Gas dynamic virtual nozzles (GDVNs) produce microscopic flow-focused liquid jets and droplets and play an important role at X-ray free-electron laser (XFEL) facilities where they are used to steer a stream of hydrated biomolecules into an X-ray focus during diffraction measurements. Highly stable and reproducible microjet and microdroplets are desired, as are flexible fabrication methods that enable integrated mixing microfluidics, droplet triggering mechanisms, laser illumination, and other customized features. In this study, we develop the use of high-resolution 3D nano-printing for the production of monolithic, asymmetric GDVN designs that are difficult to fabricate by other means. We also develop a dual-pulsed nanosecond image acquisition and analysis platform for the characterization of GDVN performance, including jet speed, length, diameter, and directionality, among others. We show that printed GDVNs can form microjets with very high degree of reproducibility, down to sub-micron diameters, and with water jet speeds beyond 170 m/s.
RESUMEN
Monoclonal antibodies provide an attractive alternative to small-molecule therapies for a wide range of diseases. Given the importance of G protein-coupled receptors (GPCRs) as pharmaceutical targets, there has been an immense interest in developing therapeutic monoclonal antibodies that act on GPCRs. Here we present the 3.0-Å resolution structure of a complex between the human 5-hydroxytryptamine 2B (5-HT2B) receptor and an antibody Fab fragment bound to the extracellular side of the receptor, determined by serial femtosecond crystallography with an X-ray free-electron laser. The antibody binds to a 3D epitope of the receptor that includes all three extracellular loops. The 5-HT2B receptor is captured in a well-defined active-like state, most likely stabilized by the crystal lattice. The structure of the complex sheds light on the mechanism of selectivity in extracellular recognition of GPCRs by monoclonal antibodies.
Asunto(s)
Fragmentos Fab de Inmunoglobulinas/química , Receptor de Serotonina 5-HT2B/química , Receptor de Serotonina 5-HT2B/inmunología , Anticuerpos Monoclonales/química , Anticuerpos Monoclonales/metabolismo , Unión Competitiva , Dominio Catalítico , Cristalografía por Rayos X , Epítopos/química , Epítopos/metabolismo , Ergotamina/química , Ergotamina/metabolismo , Humanos , Fragmentos Fab de Inmunoglobulinas/metabolismo , Modelos Moleculares , Conformación Proteica , Receptor de Serotonina 5-HT2B/metabolismo , Agonistas de Receptores de Serotonina/química , Agonistas de Receptores de Serotonina/metabolismoRESUMEN
To understand how molecules function in biological systems, new methods are required to obtain atomic resolution structures from biological material under physiological conditions. Intense femtosecond-duration pulses from X-ray free-electron lasers (XFELs) can outrun most damage processes, vastly increasing the tolerable dose before the specimen is destroyed. This in turn allows structure determination from crystals much smaller and more radiation sensitive than previously considered possible, allowing data collection from room temperature structures and avoiding structural changes due to cooling. Regardless, high-resolution structures obtained from XFEL data mostly use crystals far larger than 1 µm3 in volume, whereas the X-ray beam is often attenuated to protect the detector from damage caused by intense Bragg spots. Here, we describe the 2 Å resolution structure of native nanocrystalline granulovirus occlusion bodies (OBs) that are less than 0.016 µm3 in volume using the full power of the Linac Coherent Light Source (LCLS) and a dose up to 1.3 GGy per crystal. The crystalline shell of granulovirus OBs consists, on average, of about 9,000 unit cells, representing the smallest protein crystals to yield a high-resolution structure by X-ray crystallography to date. The XFEL structure shows little to no evidence of radiation damage and is more complete than a model determined using synchrotron data from recombinantly produced, much larger, cryocooled granulovirus granulin microcrystals. Our measurements suggest that it should be possible, under ideal experimental conditions, to obtain data from protein crystals with only 100 unit cells in volume using currently available XFELs and suggest that single-molecule imaging of individual biomolecules could almost be within reach.
Asunto(s)
Cristalografía/métodos , Electrones , Granulovirus/ultraestructura , Péptidos y Proteínas de Señalización Intercelular/química , Rayos Láser , Cristalografía/instrumentación , Granulovirus/química , Modelos Moleculares , Progranulinas , Estructura Secundaria de Proteína , SincrotronesRESUMEN
BACKGROUND: Ever since the first atomic structure of an enzyme was solved, the discovery of the mechanism and dynamics of reactions catalyzed by biomolecules has been the key goal for the understanding of the molecular processes that drive life on earth. Despite a large number of successful methods for trapping reaction intermediates, the direct observation of an ongoing reaction has been possible only in rare and exceptional cases. RESULTS: Here, we demonstrate a general method for capturing enzyme catalysis "in action" by mix-and-inject serial crystallography (MISC). Specifically, we follow the catalytic reaction of the Mycobacterium tuberculosis ß-lactamase with the third-generation antibiotic ceftriaxone by time-resolved serial femtosecond crystallography. The results reveal, in near atomic detail, antibiotic cleavage and inactivation from 30 ms to 2 s. CONCLUSIONS: MISC is a versatile and generally applicable method to investigate reactions of biological macromolecules, some of which are of immense biological significance and might be, in addition, important targets for structure-based drug design. With megahertz X-ray pulse rates expected at the Linac Coherent Light Source II and the European X-ray free-electron laser, multiple, finely spaced time delays can be collected rapidly, allowing a comprehensive description of biomolecular reactions in terms of structure and kinetics from the same set of X-ray data.
Asunto(s)
Antibacterianos/química , Proteínas Bacterianas/química , Ceftriaxona/química , Cristalografía por Rayos X/métodos , Mycobacterium tuberculosis/enzimología , beta-Lactamasas/química , Proteínas Bacterianas/genética , Biocatálisis , Resistencia a las Cefalosporinas/genética , Cinética , Rayos Láser , Modelos Moleculares , Factores de Tiempo , beta-Lactamasas/genéticaRESUMEN
We describe a method to measure ultrafast protein structural changes using time-resolved wide-angle X-ray scattering at an X-ray free-electron laser. We demonstrated this approach using multiphoton excitation of the Blastochloris viridis photosynthetic reaction center, observing an ultrafast global conformational change that arises within picoseconds and precedes the propagation of heat through the protein. This provides direct structural evidence for a 'protein quake': the hypothesis that proteins rapidly dissipate energy through quake-like structural motions.
Asunto(s)
Transferencia de Energía/efectos de la radiación , Rayos Láser , Ficobiliproteínas/efectos de la radiación , Ficobiliproteínas/ultraestructura , Dispersión del Ángulo Pequeño , Difracción de Rayos X/métodos , Ficobiliproteínas/química , Conformación Proteica/efectos de la radiación , Dosis de RadiaciónRESUMEN
X-ray crystallography provides the vast majority of macromolecular structures, but the success of the method relies on growing crystals of sufficient size. In conventional measurements, the necessary increase in X-ray dose to record data from crystals that are too small leads to extensive damage before a diffraction signal can be recorded. It is particularly challenging to obtain large, well-diffracting crystals of membrane proteins, for which fewer than 300 unique structures have been determined despite their importance in all living cells. Here we present a method for structure determination where single-crystal X-ray diffraction 'snapshots' are collected from a fully hydrated stream of nanocrystals using femtosecond pulses from a hard-X-ray free-electron laser, the Linac Coherent Light Source. We prove this concept with nanocrystals of photosystem I, one of the largest membrane protein complexes. More than 3,000,000 diffraction patterns were collected in this study, and a three-dimensional data set was assembled from individual photosystem I nanocrystals (â¼200 nm to 2 µm in size). We mitigate the problem of radiation damage in crystallography by using pulses briefer than the timescale of most damage processes. This offers a new approach to structure determination of macromolecules that do not yield crystals of sufficient size for studies using conventional radiation sources or are particularly sensitive to radiation damage.