RESUMO
The neuromodulator melatonin synchronizes circadian rhythms and related physiological functions through the actions of two G-protein-coupled receptors: MT1 and MT2. Circadian release of melatonin at night from the pineal gland activates melatonin receptors in the suprachiasmatic nucleus of the hypothalamus, synchronizing the physiology and behaviour of animals to the light-dark cycle1-4. The two receptors are established drug targets for aligning circadian phase to this cycle in disorders of sleep5,6 and depression1-4,7-9. Despite their importance, few in vivo active MT1-selective ligands have been reported2,8,10-12, hampering both the understanding of circadian biology and the development of targeted therapeutics. Here we docked more than 150 million virtual molecules to an MT1 crystal structure, prioritizing structural fit and chemical novelty. Of these compounds, 38 high-ranking molecules were synthesized and tested, revealing ligands with potencies ranging from 470 picomolar to 6 micromolar. Structure-based optimization led to two selective MT1 inverse agonists-which were topologically unrelated to previously explored chemotypes-that acted as inverse agonists in a mouse model of circadian re-entrainment. Notably, we found that these MT1-selective inverse agonists advanced the phase of the mouse circadian clock by 1.3-1.5 h when given at subjective dusk, an agonist-like effect that was eliminated in MT1- but not in MT2-knockout mice. This study illustrates the opportunities for modulating melatonin receptor biology through MT1-selective ligands and for the discovery of previously undescribed, in vivo active chemotypes from structure-based screens of diverse, ultralarge libraries.
Assuntos
Ritmo Circadiano/fisiologia , Ligantes , Receptores de Melatonina/agonistas , Receptores de Melatonina/metabolismo , Animais , Ritmo Circadiano/efeitos dos fármacos , Escuridão , Avaliação Pré-Clínica de Medicamentos , Agonismo Inverso de Drogas , Feminino , Humanos , Luz , Masculino , Camundongos , Camundongos Knockout , Simulação de Acoplamento Molecular , Receptor MT1 de Melatonina/agonistas , Receptor MT1 de Melatonina/deficiência , Receptor MT1 de Melatonina/genética , Receptor MT1 de Melatonina/metabolismo , Receptor MT2 de Melatonina/agonistas , Receptor MT2 de Melatonina/deficiência , Receptor MT2 de Melatonina/genética , Receptor MT2 de Melatonina/metabolismo , Receptores de Melatonina/deficiência , Receptores de Melatonina/genética , Bibliotecas de Moléculas Pequenas/farmacologia , Especificidade por Substrato/genéticaRESUMO
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.
Assuntos
Elétrons , Lasers , Modelos Moleculares , Receptor MT2 de Melatonina/química , Receptor MT2 de Melatonina/metabolismo , Cristalização , Diabetes Mellitus Tipo 2/genética , Humanos , Indenos/química , Indenos/metabolismo , Ligantes , Melatonina/análogos & derivados , Melatonina/química , Melatonina/metabolismo , Simulação de Acoplamento Molecular , Simulação de Dinâmica Molecular , Mutação , Receptor MT1 de Melatonina/química , Receptor MT1 de Melatonina/metabolismo , Receptor MT2 de Melatonina/genética , Relação Estrutura-Atividade , Especificidade por SubstratoRESUMO
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.
Assuntos
Elétrons , Lasers , Modelos Moleculares , Receptor MT1 de Melatonina/química , Receptor MT1 de Melatonina/metabolismo , Acetamidas/química , Acetamidas/metabolismo , Sequência de Aminoácidos , Antidepressivos/química , Antidepressivos/metabolismo , Cristalização , Humanos , Indenos/química , Indenos/metabolismo , Ligantes , Melatonina/análogos & derivados , Melatonina/química , Simulação de Acoplamento Molecular , Mutação , Receptor MT1 de Melatonina/agonistas , Receptor MT1 de Melatonina/genética , Receptor 5-HT2C de Serotonina/química , Relação Estrutura-Atividade , Especificidade por SubstratoRESUMO
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.
RESUMO
X-ray free electron lasers (XFELs) have the potential to revolutionize macromolecular structural biology due to the unique combination of spatial coherence, extreme peak brilliance, and short duration of X-ray pulses. A recently emerged serial femtosecond (fs) crystallography (SFX) approach using XFEL radiation overcomes some of the biggest hurdles of traditional crystallography related to radiation damage through the diffraction-before-destruction principle. Intense fs XFEL pulses enable high-resolution room-temperature structure determination of difficult-to-crystallize biological macromolecules, while simultaneously opening a new era of time-resolved structural studies. Here, we review the latest developments in instrumentation, sample delivery, data analysis, crystallization methods, and applications of SFX to important biological questions, and conclude with brief insights into the bright future of structural biology using XFELs.
Assuntos
Cristalografia/métodos , Elétrons , Lasers , Substâncias Macromoleculares/química , Fatores de Tempo , Raios XRESUMO
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.
Assuntos
Transferência de Energia/efeitos da radiação , Lasers , Ficobiliproteínas/efeitos da radiação , Ficobiliproteínas/ultraestrutura , Espalhamento a Baixo Ângulo , Difração de Raios X/métodos , Ficobiliproteínas/química , Conformação Proteica/efeitos da radiação , Doses de RadiaçãoRESUMO
X-ray free electron laser (X-FEL)-based serial femtosecond crystallography is an emerging method with potential to rapidly advance the challenging field of membrane protein structural biology. Here we recorded interpretable diffraction data from micrometer-sized lipidic sponge phase crystals of the Blastochloris viridis photosynthetic reaction center delivered into an X-FEL beam using a sponge phase micro-jet.
Assuntos
Cristalografia por Raios X/métodos , Bicamadas Lipídicas/química , Proteínas de Membrana/química , Proteínas de Membrana/ultraestrutura , Ligação Proteica , Conformação Proteica/efeitos da radiação , Raios XRESUMO
Time-resolved wide-angle x-ray scattering (TR-WAXS) is an emerging biophysical method which probes protein conformational changes with time. Here we present a comparative TR-WAXS study of native green-absorbing proteorhodopsin (pR) from SAR86 and a halogenated derivative for which the retinal chromophore has been replaced with 13-desmethyl-13-iodoretinal (13-I-pR). Transient absorption spectroscopy differences show that the 13-I-pR photocycle is both accelerated and displays more complex kinetics than native pR. TR-WAXS difference data also reveal that protein structural changes rise and decay an order-of-magnitude more rapidly for 13-I-pR than native pR. Despite these differences, the amplitude and nature of the observed helical motions are not significantly affected by the substitution of the retinal's C-20 methyl group with an iodine atom. Molecular dynamics simulations indicate that a significant increase in free energy is associated with the 13-cis conformation of 13-I-pR, consistent with our observation that the transient 13-I-pR conformational state is reached more rapidly. We conclude that although the conformational trajectory is accelerated, the major transient conformation of pR is unaffected by the substitution of an iodinated retinal chromophore.
Assuntos
Retinaldeído/química , Rodopsina/química , Difração de Raios X , Cor , Iodo/química , Isomerismo , Modelos Moleculares , Conformação Proteica , Rodopsinas Microbianas , Termodinâmica , Fatores de TempoRESUMO
A major current deficit in structural biology is the lack of high-resolution structures of eukaryotic membrane proteins, many of which are key drug targets for the treatment of disease. Numerous eukaryotic membrane proteins require specific lipids for their stability and activity, and efforts to crystallize and solve the structures of membrane proteins that do not address the issue of lipids frequently end in failure rather than success. To help address this problem, we have developed a sparse matrix crystallization screen consisting of 48 lipidic-sponge phase conditions. Sponge phases form liquid lipid bilayer environments which are suitable for conventional hanging- and sitting-drop crystallization experiments. Using the sponge phase screen, we obtained crystals of several different membrane proteins from bacterial and eukaryotic sources. We also demonstrate how the screen may be manipulated by incorporating specific lipids such as cholesterol; this modification led to crystals being recovered from a bacterial photosynthetic core complex.
Assuntos
Cristalização/métodos , Bicamadas Lipídicas/química , Proteínas de Membrana/ultraestrutura , Proteínas de Bactérias/química , Detergentes/química , Proteínas de Membrana/química , Espalhamento a Baixo Ângulo , Difração de Raios XRESUMO
The long-anticipated high-resolution structures of the human melatonin G protein-coupled receptors MT1 and MT2 , involved in establishing and maintaining circadian rhythm, were obtained in complex with two melatonin analogs and two approved anti-insomnia and antidepression drugs using X-ray free-electron laser serial femtosecond crystallography. The structures shed light on the overall conformation and unusual structural features of melatonin receptors, as well as their ligand binding sites and the melatonergic pharmacophore, thereby providing insights into receptor subtype selectivity. The structures revealed an occluded orthosteric ligand binding site with a membrane-buried channel for ligand entry in both receptors, and an additional putative ligand entry path in MT2 from the extracellular side. This unexpected ligand entry mode contributes to facilitating the high specificity with which melatonin receptors bind their cognate ligand and exclude structurally similar molecules such as serotonin, the biosynthetic precursor of melatonin. Finally, the MT2 structure allowed accurate mapping of type 2 diabetes-related single-nucleotide polymorphisms, where a clustering of residues in helices I and II on the protein-membrane interface was observed which could potentially influence receptor oligomerization. The role of receptor oligomerization is further discussed in light of the differential interaction of MT1 and MT2 with GPR50, a regulatory melatonin coreceptor. The melatonin receptor structures will facilitate design of selective tool compounds to further dissect the specific physiological function of each receptor subtype as well as provide a structural basis for next-generation sleeping aids and other drugs targeting these receptors with higher specificity and fewer side effects.
Assuntos
Receptores de Melatonina/química , Receptores de Melatonina/metabolismo , Animais , Desenho de Fármacos , Humanos , Conformação Proteica , Medicamentos Indutores do SonoRESUMO
Melatonin receptors MT1 and MT2 are involved in synchronizing circadian rhythms and are important targets for treating sleep and mood disorders, type-2 diabetes and cancer. Here, we performed large scale structure-based virtual screening for new ligand chemotypes using recently solved high-resolution 3D crystal structures of agonist-bound MT receptors. Experimental testing of 62 screening candidates yielded the discovery of 10 new agonist chemotypes with sub-micromolar potency at MT receptors, with compound 21 reaching EC50 of 0.36 nM. Six of these molecules displayed selectivity for MT2 over MT1. Moreover, two most potent agonists, including 21 and a close derivative of melatonin, 28, had dramatically reduced arrestin recruitment at MT2, while compound 37 was devoid of Gi signaling at MT1, implying biased signaling. This study validates the suitability of the agonist-bound orthosteric pocket in the MT receptor structures for the structure-based discovery of selective agonists.
Assuntos
Descoberta de Drogas/métodos , Receptores de Melatonina/agonistas , Sítios de Ligação , Avaliação Pré-Clínica de Medicamentos/métodos , Humanos , Receptor MT1 de Melatonina/agonistas , Receptor MT2 de Melatonina/agonistas , Relação Estrutura-AtividadeRESUMO
Membrane proteins are embedded in a lipid bilayer and maintain strong interactions with lipid molecules. Tightly bound lipids are responsible for vertical positioning and integration of proteins in the membrane and for assembly of multisubunit complexes and occasionally act as substrates. In this work we present the lipidic sponge phase crystal structure of the reaction center from Blastochloris viridis to 1.86 A, which reveals lipid molecules interacting with the protein surface. A diacylglycerol molecule is bound, through a thioether bond, to the N-terminus of the tetraheme cytochrome c subunit. From the electron density recovered at the Q(B) site and the observed change in recombination kinetics in lipidic sponge phase-grown crystals, the mobile ubiquinone appears to be displaced by a monoolein molecule. A 36 A long electron density feature is observed at the interface of transmembrane helices belonging to the H- and M-subunits, probably arising from an unidentified lipid.
Assuntos
Lipídeos/química , Complexo de Proteínas do Centro de Reação Fotossintética/química , Proteínas de Bactérias/análise , Proteínas de Bactérias/química , Proteínas de Bactérias/metabolismo , Sítios de Ligação , Cristalografia , Diglicerídeos/química , Cinética , Bicamadas Lipídicas , Modelos Moleculares , Complexo de Proteínas do Centro de Reação Fotossintética/metabolismo , Ligação Proteica , Conformação Proteica , Rhizobium/química , Ubiquinona/metabolismo , Difração de Raios XRESUMO
Rhodopsin is the G protein-coupled receptor (GPCR) that serves as a dim-light receptor for vision in vertebrates. We probed light-induced conformational changes in rhodopsin in its native membrane environment at room temperature using time-resolved wide-angle x-ray scattering. We observed a rapid conformational transition that is consistent with an outward tilt of the cytoplasmic portion of transmembrane helix 6 concomitant with an inward movement of the cytoplasmic portion of transmembrane helix 5. These movements were considerably larger than those reported from the basis of crystal structures of activated rhodopsin, implying that light activation of rhodopsin involves a more extended conformational change than was previously suggested.
Assuntos
Luz , Modelos Moleculares , Rodopsina/química , Rodopsina/efeitos da radiação , Animais , Bovinos , Conformação Proteica/efeitos da radiação , Espalhamento de RadiaçãoRESUMO
OBJECTIVE: To determine the influence of ethnic origin on the pharmacokinetic and pharmacodynamic properties of melagatran after oral administration of ximelagatran, a novel oral direct thrombin inhibitor. STUDY DESIGN: This was an open-label, non-randomised study with a single study session. SUBJECTS: Thirty-six young healthy male subjects living in France were divided equally according to their ethnic origin (African, Asian and Caucasian). METHODS: All subjects received a single 50mg oral dose of ximelagatran in solution. Blood and urine samples for pharmacokinetic evaluation were collected up to 12 and 24 hours after administration, respectively. Blood samples were also collected to determine the activated partial thromboplastin time (APTT), an ex vivo coagulation time measurement used to demonstrate inhibition of thrombin, up to 24 hours after administration. RESULTS: The absorption of ximelagatran, and its bioconversion to melagatran, was rapid in all three ethnic groups. The metabolite pattern in plasma and urine was similar in all groups, with melagatran being the dominant compound. For ximelagatran, the mean area under the plasma concentration-time curve (AUC) was similar in the three groups, suggesting that there was no difference in the extent to which ximelagatran was absorbed. Melagatran AUC was higher in the Asian subjects, with a mean Asian/Caucasian ratio (95% CI) of 1.23 (1.04, 1.45). This was presumably because of their lower bodyweight, which is correlated to lower renal function. Following normalisation for bodyweight, there were no statistically significant differences between the three ethnic groups. This finding suggests that renal elimination was lower for Asian subjects, whereas there were no differences in the conversion of ximelagatran to melagatran. The interindividual variability of melagatran AUC was low (coefficient of variation 19-26%), and the mean bioavailability of melagatran, estimated using a mean value for melagatran clearance obtained from Caucasian subjects in a previous study, was approximately 20% in all groups (range of mean values 19-23%). APTT increased nonlinearly with increasing melagatran plasma concentration, and no difference in the concentration-response relationship was observed between the groups. CONCLUSIONS: After oral administration of ximelagatran, the pharmacokinetic and pharmacodynamic properties of melagatran are independent of ethnic origin. The elimination of melagatran is correlated with renal function.
Assuntos
Azetidinas/administração & dosagem , Glicina , Glicina/análogos & derivados , Pró-Fármacos/administração & dosagem , Grupos Raciais , Trombina/antagonistas & inibidores , Administração Oral , Adulto , Área Sob a Curva , Povo Asiático , Benzilaminas , População Negra , Glicina/sangue , Glicina/farmacologia , Glicina/urina , Humanos , Inativação Metabólica/fisiologia , Masculino , População BrancaRESUMO
OBJECTIVE: To investigate the influence of age on the pharmacokinetics and pharmacodynamics of ximelagatran. STUDY DESIGN: This was an open-label, randomised, 3 x 3 crossover study with 4 study days, separated by washout periods of 7 days. SUBJECTS: Subjects comprised 6 healthy young men (aged 20-27 years) and 12 healthy older men and women (aged 56-70 years). METHODS: All subjects received a 2mg intravenous infusion of melagatran over 10 minutes followed, in randomised sequence, by a 20 mg immediate-release tablet of ximelagatran with breakfast, a 20 mg immediate-release tablet of ximelagatran while fasting, and a 7.5 mg subcutaneous injection of ximelagatran. The primary variables were the plasma concentration of melagatran, the active form of ximelagatran, and the activated partial thromboplastin time (APTT), an ex vivo coagulation time measurement used to demonstrate inhibition of thrombin. RESULTS: After oral and subcutaneous administration, ximelagatran was rapidly absorbed and biotransformed to melagatran, its active form and the dominant compound in plasma. The metabolite pattern in plasma and urine was similar in young and older subjects after both oral and subcutaneous administration of ximelagatran clearance of melagatran was correlated with renal function, resulting in about 40% (after intravenous melagatran) to 60% (after oral and subcutaneous ximelagatran) higher melagatran exposure in the older than in the young subjects. Renal clearance of melagatran, was 7.7 L/h and 4.9 L/h in the younger and older subjects, respectively. The interindividual variability inn the area under the melagatran plasma concentration-time curve was low following all regimens (coefficient variation 12-25%). The mean bioavailability of melagatran in young and older subjects was approximately 18 and 12% , respectively, following oral administration of ximalagratan, and 38 and 45%, respectively, following subcutaneous administration of ximelagatran. The bioavailability of melagatran following oral administration of ximelagatran was unaffected by whether subjects were fed or fasting, although the plasma concentration of melagatran peaked about 1 hour later under fed than fasting conditions, due to gastric emptying of the immediate-release tablet formulation used. The APTT as prolonged with increasing melagatran plasma concentration-effect relationship was independent of age. CONCLUSIONS: There were no age-dependent differences in the absorption and biotransformation of ximelagatran, and the observed differences in exposure to melagatran can be explained by differences in renal function between the young and older subjects.
Assuntos
Azetidinas/farmacologia , Fibrinolíticos/farmacologia , Glicina/análogos & derivados , Pró-Fármacos/farmacologia , Trombina/antagonistas & inibidores , Administração Oral , Adulto , Fatores Etários , Idoso , Área Sob a Curva , Azetidinas/farmacocinética , Benzilaminas , Disponibilidade Biológica , Estudos Cross-Over , Feminino , Fibrinolíticos/farmacocinética , Interações Alimento-Droga , Glicina/sangue , Glicina/farmacocinética , Glicina/urina , Humanos , Infusões Intravenosas , Injeções Subcutâneas , Masculino , Taxa de Depuração Metabólica , Pessoa de Meia-Idade , Pró-Fármacos/farmacocinéticaRESUMO
Serial femtosecond crystallography is an X-ray free-electron-laser-based method with considerable potential to have an impact on challenging problems in structural biology. Here we present X-ray diffraction data recorded from microcrystals of the Blastochloris viridis photosynthetic reaction centre to 2.8 Å resolution and determine its serial femtosecond crystallography structure to 3.5 Å resolution. Although every microcrystal is exposed to a dose of 33 MGy, no signs of X-ray-induced radiation damage are visible in this integral membrane protein structure.
Assuntos
Cristalografia por Raios X/métodos , Hyphomicrobiaceae/química , Complexo de Proteínas do Centro de Reação Fotossintética/química , Conformação ProteicaRESUMO
The Trypanosoma brucei cysteine protease cathepsin B (TbCatB), which is involved in host protein degradation, is a promising target to develop new treatments against sleeping sickness, a fatal disease caused by this protozoan parasite. The structure of the mature, active form of TbCatB has so far not provided sufficient information for the design of a safe and specific drug against T. brucei. By combining two recent innovations, in vivo crystallization and serial femtosecond crystallography, we obtained the room-temperature 2.1 angstrom resolution structure of the fully glycosylated precursor complex of TbCatB. The structure reveals the mechanism of native TbCatB inhibition and demonstrates that new biomolecular information can be obtained by the "diffraction-before-destruction" approach of x-ray free-electron lasers from hundreds of thousands of individual microcrystals.
Assuntos
Catepsina B/química , Proteínas de Protozoários/química , Trypanosoma brucei brucei/enzimologia , Sequência de Aminoácidos , Animais , Domínio Catalítico , Catepsina B/antagonistas & inibidores , Cristalização , Cristalografia por Raios X , Precursores Enzimáticos/química , Glicosilação , Modelos Moleculares , Dados de Sequência Molecular , Conformação Proteica , Proteínas de Protozoários/antagonistas & inibidores , Células Sf9 , Spodoptera , Raios XRESUMO
Structure determination of proteins and other macromolecules has historically required the growth of high-quality crystals sufficiently large to diffract x-rays efficiently while withstanding radiation damage. We applied serial femtosecond crystallography (SFX) using an x-ray free-electron laser (XFEL) to obtain high-resolution structural information from microcrystals (less than 1 micrometer by 1 micrometer by 3 micrometers) of the well-characterized model protein lysozyme. The agreement with synchrotron data demonstrates the immediate relevance of SFX for analyzing the structure of the large group of difficult-to-crystallize molecules.
Assuntos
Cristalografia por Raios X/métodos , Conformação Proteica , Animais , Lasers , Muramidase/química , Muramidase/efeitos da radiaçãoRESUMO
Photosynthetic reaction centers convert the energy content of light into a transmembrane potential difference and so provide the major pathway for energy input into the biosphere. We applied time-resolved Laue diffraction to study light-induced conformational changes in the photosynthetic reaction center complex of Blastochloris viridis. The side chain of TyrL162, which lies adjacent to the special pair of bacteriochlorophyll molecules that are photooxidized in the primary light conversion event of photosynthesis, was observed to move 1.3 angstroms closer to the special pair after photoactivation. Free energy calculations suggest that this movement results from the deprotonation of this conserved tyrosine residue and provides a mechanism for stabilizing the primary charge separation reactions of photosynthesis.
Assuntos
Proteínas de Bactérias/química , Hyphomicrobiaceae/química , Luz , Complexo de Proteínas do Centro de Reação Fotossintética/química , Proteínas de Bactérias/metabolismo , Bacterioclorofilas/química , Bacterioclorofilas/metabolismo , Cristalografia por Raios X , Citocromos c/química , Citocromos c/metabolismo , Transporte de Elétrons , Ligação de Hidrogênio , Concentração de Íons de Hidrogênio , Hyphomicrobiaceae/metabolismo , Modelos Moleculares , Simulação de Dinâmica Molecular , Oxirredução , Complexo de Proteínas do Centro de Reação Fotossintética/metabolismo , Conformação Proteica , Prótons , Quinonas/química , Quinonas/metabolismo , TermodinâmicaRESUMO
Membrane protein structural biology is enjoying a steady acceleration in the rate of success. Nevertheless, numerous membrane protein targets are resistant to the traditional approach of directly crystallizing detergent solubilized and purified protein and the 'niche market' of lipidic phase crystallization is emerging as a powerful complement. These approaches, including lipidic cubic phase, lipidic sponge phase, and bicelle crystallization methods, all immerse purified membrane protein within a lipid rich matrix before crystallization. This environment is hypothesized to contribute to the protein's long-term structural stability and thereby favor crystallization. Spectacular recent successes include the high-resolution structures of the beta(2)-adrenergic G-protein-coupled receptor, the A(2A) adenosine G-protein-coupled receptor, and the mitochondrial voltage dependent anion channel. In combination with technical innovations aiming to popularize these methods, lipidic phase crystallization approaches can be expected to deliver an increasing scientific impact as the field develops.