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1.
Nat Commun ; 11(1): 996, 2020 02 21.
Artigo em Inglês | MEDLINE | ID: mdl-32081905

RESUMO

Serial X-ray crystallography at free-electron lasers allows to solve biomolecular structures from sub-micron-sized crystals. However, beam time at these facilities is scarce, and involved sample delivery techniques are required. On the other hand, rotation electron diffraction (MicroED) has shown great potential as an alternative means for protein nano-crystallography. Here, we present a method for serial electron diffraction of protein nanocrystals combining the benefits of both approaches. In a scanning transmission electron microscope, crystals randomly dispersed on a sample grid are automatically mapped, and a diffraction pattern at fixed orientation is recorded from each at a high acquisition rate. Dose fractionation ensures minimal radiation damage effects. We demonstrate the method by solving the structure of granulovirus occlusion bodies and lysozyme to resolutions of 1.55 Å and 1.80 Å, respectively. Our method promises to provide rapid structure determination for many classes of materials with minimal sample consumption, using readily available instrumentation.


Assuntos
Cristalografia/métodos , Proteínas/química , Microscopia Eletrônica de Transmissão e Varredura , Modelos Moleculares , Muramidase/química , Muramidase/ultraestrutura , Nanopartículas/química , Nanopartículas/ultraestrutura , Proteínas de Matriz de Corpos de Inclusão/química , Proteínas de Matriz de Corpos de Inclusão/ultraestrutura , Tamanho da Partícula , Conformação Proteica , Proteínas/ultraestrutura
2.
Proc Natl Acad Sci U S A ; 117(8): 4071-4077, 2020 02 25.
Artigo em Inglês | MEDLINE | ID: mdl-32041886

RESUMO

Copper-containing nitrite reductases (CuNIRs) transform nitrite to gaseous nitric oxide, which is a key process in the global nitrogen cycle. The catalytic mechanism has been extensively studied to ultimately achieve rational control of this important geobiochemical reaction. However, accumulated structural biology data show discrepancies with spectroscopic and computational studies; hence, the reaction mechanism is still controversial. In particular, the details of the proton transfer involved in it are largely unknown. This situation arises from the failure of determining positions of hydrogen atoms and protons, which play essential roles at the catalytic site of CuNIRs, even with atomic resolution X-ray crystallography. Here, we determined the 1.50 Šresolution neutron structure of a CuNIR from Geobacillus thermodenitrificans (trimer molecular mass of ∼106 kDa) in its resting state at low pH. Our neutron structure reveals the protonation states of catalytic residues (deprotonated aspartate and protonated histidine), thus providing insights into the catalytic mechanism. We found that a hydroxide ion can exist as a ligand to the catalytic Cu atom in the resting state even at a low pH. This OH-bound Cu site is unexpected from previously given X-ray structures but consistent with a reaction intermediate suggested by computational chemistry. Furthermore, the hydrogen-deuterium exchange ratio in our neutron structure suggests that the intramolecular electron transfer pathway has a hydrogen-bond jump, which is proposed by quantum chemistry. Our study can seamlessly link the structural biology to the computational chemistry of CuNIRs, boosting our understanding of the enzymes at the atomic and electronic levels.


Assuntos
Cobre/química , Cristalografia/métodos , Geobacillus/enzimologia , Nitrito Redutases/química , Nitrito Redutases/metabolismo , Domínio Catalítico , Cristalização , Regulação Bacteriana da Expressão Gênica/fisiologia , Regulação Enzimológica da Expressão Gênica/fisiologia , Geobacillus/genética , Geobacillus/metabolismo , Modelos Moleculares , Nitrito Redutases/genética , Conformação Proteica
3.
Acta Crystallogr D Struct Biol ; 76(Pt 1): 1-8, 2020 Jan 01.
Artigo em Inglês | MEDLINE | ID: mdl-31909738

RESUMO

The conventional approach to search-model identification in molecular replacement (MR) is to screen a database of known structures using the target sequence. However, this strategy is not always effective, for example when the relationship between sequence and structural similarity fails or when the crystal contents are not those expected. An alternative approach is to identify suitable search models directly from the experimental data. SIMBAD is a sequence-independent MR pipeline that uses either a crystal lattice search or MR functions to directly locate suitable search models from databases. The previous version of SIMBAD used the fast AMoRe rotation-function search. Here, a new version of SIMBAD which makes use of Phaser and its likelihood scoring to improve the sensitivity of the pipeline is presented. It is shown that the additional compute time potentially required by the more sophisticated scoring is counterbalanced by the greater sensitivity, allowing more cases to trigger early-termination criteria, rather than running to completion. Using Phaser solved 17 out of 25 test cases in comparison to the ten solved with AMoRe, and it is shown that use of ensemble search models produces additional performance benefits.


Assuntos
Modelos Moleculares , Proteínas/química , Software , Cristalografia/métodos , Bases de Dados de Proteínas , Conformação Proteica
4.
Dent Mater J ; 39(2): 295-301, 2020 Mar 31.
Artigo em Inglês | MEDLINE | ID: mdl-31827055

RESUMO

The mechanical properties of highly translucent partially stabilized zirconia (PSZ) need to be improved; however, improvement of mechanical properties often decreases translucency. To overcome this problem, a monoclinic ZrO2 (mZrO2)/SiO2 dispersion was prepared and applied as a coating material for PSZ. The influence of surface treatment by the mZrO2/SiO2 dispersion on the surface topography, crystallography, and mechanical properties of highly translucent PSZ was investigated in this study. Following the treatment, the mechanical strength of highly translucent PSZ improved by 170% compared with control, for the best mZrO2/SiO2 dispersion ratio and heating temperature condition, while maintaining its translucency. The proposed coating is promising for improving the mechanical properties of highly translucent PSZ.


Assuntos
Dióxido de Silício , Zircônio , Cerâmica , Cristalografia , Materiais Dentários , Teste de Materiais , Propriedades de Superfície
5.
Proc Natl Acad Sci U S A ; 117(1): 300-307, 2020 01 07.
Artigo em Inglês | MEDLINE | ID: mdl-31852825

RESUMO

A major barrier to defining the structural intermediates that arise during the reversible photointerconversion of phytochromes between their biologically inactive and active states has been the lack of crystals that faithfully undergo this transition within the crystal lattice. Here, we describe a crystalline form of the cyclic GMP phosphodiesterases/adenylyl cyclase/FhlA (GAF) domain from the cyanobacteriochrome PixJ in Thermosynechococcus elongatus assembled with phycocyanobilin that permits reversible photoconversion between the blue light-absorbing Pb and green light-absorbing Pg states, as well as thermal reversion of Pg back to Pb. The X-ray crystallographic structure of Pb matches previous models, including autocatalytic conversion of phycocyanobilin to phycoviolobilin upon binding and its tandem thioether linkage to the GAF domain. Cryocrystallography at 150 K, which compared diffraction data from a single crystal as Pb or after irradiation with blue light, detected photoconversion product(s) based on Fobs - Fobs difference maps that were consistent with rotation of the bonds connecting pyrrole rings C and D. Further spectroscopic analyses showed that phycoviolobilin is susceptible to X-ray radiation damage, especially as Pg, during single-crystal X-ray diffraction analyses, which could complicate fine mapping of the various intermediate states. Fortunately, we found that PixJ crystals are amenable to serial femtosecond crystallography (SFX) analyses using X-ray free-electron lasers (XFELs). As proof of principle, we solved by room temperature SFX the GAF domain structure of Pb to 1.55-Å resolution, which was strongly congruent with synchrotron-based models. Analysis of these crystals by SFX should now enable structural characterization of the early events that drive phytochrome photoconversion.


Assuntos
Ficobilinas/metabolismo , Ficocianina/metabolismo , Fitocromo/química , Fitocromo/efeitos da radiação , Adenilil Ciclases/química , Adenilil Ciclases/metabolismo , Cristalografia , Cristalografia por Raios X , Cianobactérias/química , GMP Cíclico , Luz , Modelos Moleculares , Diester Fosfórico Hidrolases/química , Diester Fosfórico Hidrolases/metabolismo , Células Fotorreceptoras/metabolismo , Ficobilinas/química , Ficocianina/química , Conformação Proteica , Domínios Proteicos , Transativadores/química
6.
RNA ; 26(3): 278-289, 2020 03.
Artigo em Inglês | MEDLINE | ID: mdl-31848215

RESUMO

Ubiquitous across all domains of life, tRNAs constitute an essential component of cellular physiology, carry out an indispensable role in protein synthesis, and have been historically the subject of a wide range of biochemical and biophysical studies as prototypical folded RNA molecules. Although conformational flexibility is a well-established characteristic of tRNA structure, it is typically regarded as an adaptive property exhibited in response to an inducing event, such as the binding of a tRNA synthetase or the accommodation of an aminoacyl-tRNA into the ribosome. In this study, we present crystallographic data of a tRNA molecule to expand on this paradigm by showing that structural flexibility and plasticity are intrinsic properties of tRNAs, apparent even in the absence of other factors. Based on two closely related conformations observed within the same crystal, we posit that unbound tRNAs by themselves are flexible and dynamic molecules. Furthermore, we demonstrate that the formation of the T-loop conformation by the tRNA TΨC stem-loop, a well-characterized and classic RNA structural motif, is possible even in the absence of important interactions observed in fully folded tRNAs.


Assuntos
Conformação de Ácido Nucleico , Aminoacil-RNA de Transferência/ultraestrutura , RNA de Transferência/ultraestrutura , Anticódon/química , Anticódon/genética , Cristalografia , Escherichia coli/química , Escherichia coli/ultraestrutura , Motivos de Nucleotídeos/genética , RNA de Transferência/química , Aminoacil-RNA de Transferência/química , Ribossomos/genética , Ribossomos/ultraestrutura
7.
Cell Host Microbe ; 26(6): 739-747.e4, 2019 12 11.
Artigo em Inglês | MEDLINE | ID: mdl-31830442

RESUMO

Primate lentiviruses encode a Vif protein that counteracts the host antiviral APOBEC3 (A3) family members. The adaptation of Vif to species-specific A3 determinants is a critical event that allowed the spillover of a lentivirus from monkey reservoirs to chimpanzees and subsequently to humans, which gave rise to HIV-1 and the acquired immune deficiency syndrome (AIDS) pandemic. How Vif-A3 protein interactions are remodeled during evolution is unclear. Here, we report a 2.94 Å crystal structure of the Vif substrate receptor complex from simian immunodeficiency virus isolated from red-capped mangabey (SIVrcm). The structure of the SIVrcm Vif complex illuminates the stage of lentiviral Vif evolution that is immediately prior to entering hominid primates. Structure-function studies reveal the adaptations that allowed SIVrcm Vif to antagonize hominid A3G. These studies show a partitioning between an evolutionarily dynamic specificity determinant and a conserved protein interacting surface on Vif that enables adaptation while maintaining protein interactions required for potent A3 antagonism.


Assuntos
Produtos do Gene vif , Vírus da Imunodeficiência Símia , Desaminase APOBEC-3G/metabolismo , Síndrome de Imunodeficiência Adquirida , Animais , Cercocebus , Cristalografia , Evolução Molecular , Produtos do Gene vif/química , Produtos do Gene vif/genética , HIV-1/genética , HIV-1/metabolismo , Hominidae , Interações Hospedeiro-Patógeno , Humanos , Lentivirus/genética , Lentivirus/metabolismo , Doenças dos Macacos/virologia , Pan troglodytes , Primatas , Vírus da Imunodeficiência Símia/genética , Vírus da Imunodeficiência Símia/metabolismo , Produtos do Gene vif do Vírus da Imunodeficiência Humana/química , Produtos do Gene vif do Vírus da Imunodeficiência Humana/genética , Produtos do Gene vif do Vírus da Imunodeficiência Humana/ultraestrutura
8.
Molecules ; 25(1)2019 Dec 19.
Artigo em Inglês | MEDLINE | ID: mdl-31861563

RESUMO

This paper mainly focuses on the application of nanostructured MoO3 materials in both energy and environmental catalysis fields. MoO3 has wide tunability in bandgap, a unique semiconducting structure, and multiple valence states. Due to the natural advantage, it can be used as a high-activity metal oxide catalyst, can serve as an excellent support material, and provide opportunities to replace noble metal catalysts, thus having broad application prospects in catalysis. Herein, we comprehensively summarize the crystal structure and properties of nanostructured MoO3 and highlight the recent significant research advancements in energy and environmental catalysis. Several current challenges and perspective research directions based on nanostructured MoO3 are also discussed.


Assuntos
Molibdênio/química , Óxidos/química , Catálise , Cristalografia , Estrutura Molecular , Nanoestruturas , Processos Fotoquímicos , Água/química
9.
Chin J Nat Med ; 17(12): 906-911, 2019 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-31882044

RESUMO

A pair of new tirucallane triterpenoid epimers, picraquassins M and N (1> and 2), were isolated from the stems of Picrasma quassioides (D. Don) Benn. Their structures were determined based on comprehensive spectroscopic and X-ray crystallographic analyses. In addition, their AChE inhibitory activity, cytotoxicity against five human tumour cell lines (SW480, MCF-7, HepG2, Hela, and PANC-1), and antimicrobial activity against two bacteria (Staphylococcus. aureus 209P and Escherichia coli ATCC0111) and two fungi (Candida albicans FIM709 and Aspergillus niger R330) were evaluated.


Assuntos
Anti-Infecciosos/química , Picrasma/química , Caules de Planta/química , Triterpenos/química , Anti-Infecciosos/isolamento & purificação , Aspergillus niger/efeitos dos fármacos , Candida albicans/efeitos dos fármacos , Linhagem Celular Tumoral , China , Cristalografia , Escherichia coli/efeitos dos fármacos , Humanos , Espectroscopia de Ressonância Magnética , Estrutura Molecular , Staphylococcus aureus/efeitos dos fármacos , Triterpenos/isolamento & purificação
10.
Proc Natl Acad Sci U S A ; 116(51): 25583-25590, 2019 12 17.
Artigo em Inglês | MEDLINE | ID: mdl-31776258

RESUMO

Methylotrophy, the ability of microorganisms to grow on reduced one-carbon substrates such as methane or methanol, is a feature of various bacterial species. The prevailing oxidation pathway depends on tetrahydromethanopterin (H4MPT) and methylofuran (MYFR), an analog of methanofuran from methanogenic archaea. Formyltransferase/hydrolase complex (Fhc) generates formate from formyl-H4MPT in two consecutive reactions where MYFR acts as a carrier of one-carbon units. Recently, we chemically characterized MYFR from the model methylotroph Methylorubrum extorquens and identified an unusually long polyglutamate side chain of up to 24 glutamates. Here, we report on the crystal structure of Fhc to investigate the function of the polyglutamate side chain in MYFR and the relatedness of the enzyme complex with the orthologous enzymes in archaea. We identified MYFR as a prosthetic group that is tightly, but noncovalently, bound to Fhc. Surprisingly, the structure of Fhc together with MYFR revealed that the polyglutamate side chain of MYFR is branched and contains glutamates with amide bonds at both their α- and γ-carboxyl groups. This negatively charged and branched polyglutamate side chain interacts with a cluster of conserved positively charged residues of Fhc, allowing for strong interactions. The MYFR binding site is located equidistantly from the active site of the formyltransferase (FhcD) and metallo-hydrolase (FhcA). The polyglutamate serves therefore an additional function as a swinging linker to shuttle the one-carbon carrying amine between the two active sites, thereby likely increasing overall catalysis while decreasing the need for high intracellular MYFR concentrations.


Assuntos
Proteínas de Bactérias , Furanos , Hidroximetil e Formil Transferases , Metano , Proteínas de Bactérias/química , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Coenzimas/química , Coenzimas/metabolismo , Cristalografia , Formiatos/química , Formiatos/metabolismo , Furanos/química , Furanos/metabolismo , Hidroximetil e Formil Transferases/química , Hidroximetil e Formil Transferases/genética , Hidroximetil e Formil Transferases/metabolismo , Metano/química , Metano/metabolismo , Metanol/química , Metanol/metabolismo , Methylobacterium extorquens/enzimologia , Methylobacterium extorquens/genética , Ácido Poliglutâmico/química , Ácido Poliglutâmico/metabolismo
11.
Nat Commun ; 10(1): 5021, 2019 11 04.
Artigo em Inglês | MEDLINE | ID: mdl-31685819

RESUMO

The world's first superconducting megahertz repetition rate hard X-ray free-electron laser (XFEL), the European XFEL, began operation in 2017, featuring a unique pulse train structure with 886 ns between pulses. With its rapid pulse rate, the European XFEL may alleviate some of the increasing demand for XFEL beamtime, particularly for membrane protein serial femtosecond crystallography (SFX), leveraging orders-of-magnitude faster data collection. Here, we report the first membrane protein megahertz SFX experiment, where we determined a 2.9 Å-resolution SFX structure of the large membrane protein complex, Photosystem I, a > 1 MDa complex containing 36 protein subunits and 381 cofactors. We address challenges to megahertz SFX for membrane protein complexes, including growth of large quantities of crystals and the large molecular and unit cell size that influence data collection and analysis. The results imply that megahertz crystallography could have an important impact on structure determination of large protein complexes with XFELs.


Assuntos
Elétrons , Lasers , Proteínas de Membrana/química , Cristalografia , Cianobactérias/metabolismo , Modelos Moleculares , Complexo de Proteína do Fotossistema I/química , Complexo de Proteína do Fotossistema I/isolamento & purificação , Eletricidade Estática , Síncrotrons , Raios X
12.
Annu Rev Virol ; 6(1): 161-176, 2019 09 29.
Artigo em Inglês | MEDLINE | ID: mdl-31567066

RESUMO

Until recently X-ray crystallography has been the standard technique for virus structure determinations. Available X-ray sources have continuously improved over the decades, leading to the realization of X-ray free-electron lasers (XFELs). They provide high-intensity femtosecond X-ray pulses, which allow for new kinds of experiments by making use of the diffraction-before-destruction principle. By overcoming classical dose constraints, they at least in principle allow researchers to perform X-ray virus structure determination for single particles at room temperature. Simultaneously, the availability of XFELs led to the development of the method of serial femtosecond crystallography, where a crystal structure is determined from the measurement of hundreds to thousands of microcrystals. In the case of virus crystallography this method does not require freezing of the crystals and allows researchers to perform experiments under non-equilibrium conditions (e.g., by laser-induced temperature jumps or rapid chemical mixing), which is currently not possible with electron microscopy.


Assuntos
Cristalografia/métodos , Elétrons , Lasers , Imagem Molecular/métodos , Vírus/química , Cristalografia/instrumentação , Imagem Molecular/instrumentação , Imagem Individual de Molécula/instrumentação , Imagem Individual de Molécula/métodos , Vírus/ultraestrutura , Raios X
13.
Nat Commun ; 10(1): 4910, 2019 10 28.
Artigo em Inglês | MEDLINE | ID: mdl-31659163

RESUMO

AspH is an endoplasmic reticulum (ER) membrane-anchored 2-oxoglutarate oxygenase whose C-terminal oxygenase and tetratricopeptide repeat (TPR) domains present in the ER lumen. AspH catalyses hydroxylation of asparaginyl- and aspartyl-residues in epidermal growth factor-like domains (EGFDs). Here we report crystal structures of human AspH, with and without substrate, that reveal substantial conformational changes of the oxygenase and TPR domains during substrate binding. Fe(II)-binding by AspH is unusual, employing only two Fe(II)-binding ligands (His679/His725). Most EGFD structures adopt an established fold with a conserved Cys1-3, 2-4, 5-6 disulfide bonding pattern; an unexpected Cys3-4 disulfide bonding pattern is observed in AspH-EGFD substrate complexes, the catalytic relevance of which is supported by studies involving stable cyclic peptide substrate analogues and by effects of Ca(II) ions on activity. The results have implications for EGFD disulfide pattern processing in the ER and will enable medicinal chemistry efforts targeting human 2OG oxygenases.


Assuntos
Proteínas de Ligação ao Cálcio/química , Proteínas de Membrana/química , Oxigenases de Função Mista/química , Proteínas Musculares/química , Sequência de Aminoácidos , Asparagina/metabolismo , Proteínas de Ligação ao Cálcio/genética , Proteínas de Ligação ao Cálcio/metabolismo , Domínio Catalítico , Cristalografia , Dissulfetos/química , Dissulfetos/metabolismo , Fator de Crescimento Epidérmico/metabolismo , Compostos Ferrosos/química , Compostos Ferrosos/metabolismo , Humanos , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Oxigenases de Função Mista/genética , Oxigenases de Função Mista/metabolismo , Proteínas Musculares/genética , Proteínas Musculares/metabolismo , Conformação Proteica
14.
Nat Methods ; 16(10): 979-982, 2019 10.
Artigo em Inglês | MEDLINE | ID: mdl-31527838

RESUMO

We introduce a liquid application method for time-resolved analyses (LAMA), an in situ mixing approach for serial crystallography. Picoliter-sized droplets are shot onto chip-mounted protein crystals, achieving near-full ligand occupancy within theoretical diffusion times. We demonstrate proof-of-principle binding of GlcNac to lysozyme, and resolve glucose binding and subsequent ring opening in a time-resolved study of xylose isomerase.


Assuntos
Cristalografia/métodos , Síncrotrons , Acetilglucosamina/química , Aldose-Cetose Isomerases/química , Glucose/química , Muramidase/química , Estudo de Prova de Conceito
15.
Mater Sci Eng C Mater Biol Appl ; 104: 109966, 2019 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-31499942

RESUMO

In this study we present the first crystal structure model for bone apatite based on the analysis of individual nanocrystals by high resolution transmission electron microscopy (HRTEM). Crystallographic image processing of the obtained HRTEM images from different projections indicates symmetry reduction with respect to P63/m stoichiometric apatites and the presence of threefold symmetry along the c axis. Based on HRTEM observations and the measured Ca/P = 2 ratio we propose a structural model with phosphate-to-carbonate substitution and O vacancies localized along c axis, which explains the observed loss of 63 screw axis parallel, and the shift of mirror plane perpendicular to the c axis. Also, the presence of non-equivalent (010) surfaces has been proven. These results on the atomic structure of bone apatite nanocrystals contribute to the understanding of their biochemically controlled nucleation processes.


Assuntos
Apatitas/química , Osso e Ossos/química , Nanopartículas/química , Carbonatos/química , Cristalografia/métodos , Microscopia Eletrônica de Transmissão/métodos
16.
Appl Microbiol Biotechnol ; 103(23-24): 9543-9553, 2019 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-31482280

RESUMO

Aliphatic ketones, such as 2-butanone and 3-hexanone, with only one carbon difference among side chains adjacent to the carbonyl carbon are difficult to be reduced enantioselectively. In this study, we utilized an acetophenone reductase from Geotrichum candidum NBRC 4597 (GcAPRD) to reduce challenging aliphatic ketones such as 2-butanone (methyl ethyl ketone) and 3-hexanone (ethyl propyl ketone) to their corresponding (S)-alcohols with 94% ee and > 99% ee, respectively. Through crystallographic structure determination, it was suggested that residue Trp288 limit the size of the small binding pocket. Docking simulations imply that Trp288 plays an important role to form a C-H⋯π interaction for proper orientation of ketones in the pro-S binding pose in order to produce (S)-alcohols. The excellent (S)-enantioselectivity is due to a non-productive pro-R binding pose, consistent with the observation that the (R)-alcohol acts as an inhibitor of (S)-alcohol oxidation.


Assuntos
Oxirredutases do Álcool/química , Carbono/química , Cetonas/química , Oxirredutases/química , Sítios de Ligação , Cristalografia , Geotrichum/enzimologia , Cinética , Simulação de Acoplamento Molecular , Oxirredução , Conformação Proteica , Estereoisomerismo , Especificidade por Substrato
17.
Nat Commun ; 10(1): 3177, 2019 07 18.
Artigo em Inglês | MEDLINE | ID: mdl-31320619

RESUMO

Bacteriorhodopsin (bR) is a light-driven proton pump. The primary photochemical event upon light absorption is isomerization of the retinal chromophore. Here we used time-resolved crystallography at an X-ray free-electron laser to follow the structural changes in multiphoton-excited bR from 250 femtoseconds to 10 picoseconds. Quantum chemistry and ultrafast spectroscopy were used to identify a sequential two-photon absorption process, leading to excitation of a tryptophan residue flanking the retinal chromophore, as a first manifestation of multiphoton effects. We resolve distinct stages in the structural dynamics of the all-trans retinal in photoexcited bR to a highly twisted 13-cis conformation. Other active site sub-picosecond rearrangements include correlated vibrational motions of the electronically excited retinal chromophore, the surrounding amino acids and water molecules as well as their hydrogen bonding network. These results show that this extended photo-active network forms an electronically and vibrationally coupled system in bR, and most likely in all retinal proteins.


Assuntos
Bacteriorodopsinas/química , Halobacterium salinarum/metabolismo , Retinaldeído/química , Cristalografia , Isomerismo , Luz , Fótons , Conformação Proteica , Análise Espectral , Água/química
18.
Commun Biol ; 2: 240, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31263784

RESUMO

Mutations of human BEST1, encoding a Ca2+-activated Cl- channel (hBest1), cause macular degenerative disorders. Best1 homolog structures reveal an evolutionarily conserved channel architecture highlighted by two landmark restrictions (named the "neck" and "aperture", respectively) in the ion conducting pathway, suggesting a unique dual-switch gating mechanism, which, however, has not been characterized well. Using patch clamp and crystallography, we demonstrate that both the neck and aperture in hBest1 are Ca2+-dependent gates essential for preventing channel leakage resulting from Ca2+-independent, spontaneous gate opening. Importantly, three patient-derived mutations (D203A, I205T and Y236C) lead to Ca2+-independent leakage and elevated Ca2+-dependent anion currents due to enhanced opening of the gates. Moreover, we identify a network of residues critically involved in gate operation. Together, our results suggest an indispensable role of the neck and aperture of hBest1 for channel gating, and uncover disease-causing mechanisms of hBest1 gain-of-function mutations.


Assuntos
Bestrofinas/fisiologia , Cálcio/metabolismo , Canais de Cloreto/fisiologia , Mutação com Ganho de Função , Ativação do Canal Iônico/fisiologia , Bestrofinas/química , Cristalografia , Células HEK293 , Humanos , Técnicas de Patch-Clamp , Relação Estrutura-Atividade
19.
Biochim Biophys Acta Gene Regul Mech ; 1862(11-12): 194390, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31202783

RESUMO

Spliceosomal introns and self-splicing group II introns share a common mechanism of intron splicing where two sequential transesterification reactions remove intron lariats and ligate exons. The recent revolution in cryo-electron microscopy (cryo-EM) has allowed visualization of the spliceosome's ribozyme core. Comparison of these cryo-EM structures to recent group II intron crystal structures presents an opportunity to draw parallels between the RNA active site, substrate positioning, and product formation in these two model systems of intron splicing. In addition to shared RNA architectural features, structural similarity between group II intron encoded proteins (IEPs) and the integral spliceosomal protein Prp8 further support a shared catalytic core. These mechanistic and structural similarities support the long-held assertion that group II introns and the eukaryotic spliceosome have a common evolutionary origin. In this review, we discuss how recent structural insights into group II introns and the spliceosome facilitate the chemistry of splicing, highlight similarities between the two systems, and discuss their likely evolutionary connections. This article is part of a Special Issue entitled: RNA structure and splicing regulation edited by Francisco Baralle, Ravindra Singh and Stefan Stamm.


Assuntos
Eucariotos/citologia , RNA Catalítico/química , Spliceossomos/química , Animais , Microscopia Crioeletrônica , Cristalografia , Eucariotos/genética , Evolução Molecular , Humanos , Íntrons , Modelos Moleculares , Conformação de Ácido Nucleico , Processamento de RNA , RNA Catalítico/genética , Spliceossomos/genética
20.
Expert Opin Drug Discov ; 14(9): 933-945, 2019 09.
Artigo em Inglês | MEDLINE | ID: mdl-31184514

RESUMO

Introduction: X-ray crystallography has made important contributions to modern drug development but its application to many important drug targets has been extremely challenging. The recent emergence of X-ray free electron lasers (XFELs) and advancements in serial femtosecond crystallography (SFX) have offered new opportunities to overcome limitations of traditional crystallography to accelerate the structure-based drug discovery (SBDD) process. Areas covered: In this review, the authors describe the general principles of X-ray generation and the main properties of XFEL beams, outline details of SFX data collection and processing, and summarize the progress in the development of associated instrumentation for sample delivery and X-ray detection. An overview of the SFX applications to various important drug targets such as membrane proteins is also provided. Expert opinion: While SFX has already made clear advancements toward the understanding of the structure and dynamics of several major drug targets, its robust application in SBDD still needs further developments of new high-throughput techniques for sample production, automation of crystal delivery and data collection, as well as for processing and storage of large amounts of data. The expansion of the available XFEL beamtime is a key to the success of SFX in SBDD.


Assuntos
Cristalografia/métodos , Descoberta de Drogas/métodos , Lasers , Animais , Cristalografia por Raios X/métodos , Desenvolvimento de Medicamentos/métodos , Ensaios de Triagem em Larga Escala , Humanos , Relação Estrutura-Atividade
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