RESUMO
For serial femtosecond crystallography at X-ray free-electron lasers, which entails collection of single-pulse diffraction patterns from a constantly refreshed supply of microcrystalline sample, delivery of the sample into the X-ray beam path while maintaining low background remains a technical challenge for some experiments, especially where this methodology is applied to relatively low-ordered samples or those difficult to purify and crystallize in large quantities. This work demonstrates a scheme to encapsulate biological samples using polymer thin films and graphene to maintain sample hydration in vacuum conditions. The encapsulated sample is delivered into the X-ray beam on fixed targets for rapid scanning using the Roadrunner fixed-target system towards a long-term goal of low-background measurements on weakly diffracting samples. As a proof of principle, we used microcrystals of the 24â kDa rapid encystment protein (REP24) to provide a benchmark for polymer/graphene sandwich performance. The REP24 microcrystal unit cell obtained from our sandwiched in-vacuum sample was consistent with previously established unit-cell parameters and with those measured by us without encapsulation in humidified helium, indicating that the platform is robust against evaporative losses. While significant scattering from water was observed because of the sample-deposition method, the polymer/graphene sandwich itself was shown to contribute minimally to background scattering.
RESUMO
Nanolipoprotein particles (NLPs), also called "nanodiscs", are discoidal particles with a patch of lipid bilayer corralled by apolipoproteins. NLPs have long been of interest due to both their utility as membrane-model systems into which membrane proteins can be inserted and solubilized and their physiological role in lipid and cholesterol transport via HDL and LDL maturation, which are important for human health. Serial femtosecond crystallography (SFX) at X-ray free electron lasers (XFELs) is a powerful approach for structural biology of membrane proteins, which are traditionally difficult to crystallize as large single crystals capable of producing high-quality diffraction suitable for structure determination. To facilitate understanding of the specific role of two apolipoprotein/lipid complexes, ApoA1 and ApoE4, in lipid binding and HDL/LDL particle maturation dynamics and develop new SFX methods involving NLP membrane protein encapsulation, we have prepared and crystallized homogeneous populations of ApoA1 and ApoE4 NLPs. Crystallization of empty NLPs yields semi-ordered objects that appear crystalline and give highly anisotropic and diffuse X-ray diffraction, similar in characteristics to fiber diffraction. Several unit cell parameters were approximately determined for both NLPs from these measurements. Thus, low-background, sample conservative methods of delivery are critical. Here we implemented a fixed target sample delivery scheme utilizing the Roadrunner fast-scanning system and ultra-thin polymer/graphene support films, providing a low-volume, low-background approach to membrane protein SFX. This study represents initial steps in obtaining structural information for ApoA1 and ApoE4 NLPs and developing this system as a supporting scaffold for future structural studies of membrane proteins crystalized in a native lipid environment.
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Tuberculosis (TB) infects one-third of the world population. Despite 50 years of available drug treatments, TB continues to increase at a significant rate. The failure to control TB stems in part from the expense of delivering treatment to infected individuals and from complex treatment regimens. Incomplete treatment has fueled the emergence of multi-drug resistant (MDR) strains of Mycobacterium tuberculosis (Mtb). Reducing non-compliance by reducing the duration of chemotherapy will have a great impact on TB control. The development of new drugs that either kill persisting organisms, inhibit bacilli from entering the persistent phase, or convert the persistent bacilli into actively growing cells susceptible to our current drugs will have a positive effect. We are taking a multidisciplinary approach that will identify and characterize new drug targets that are essential for persistent Mtb. Targets are exposed to a battery of analyses including microarray experiments, bioinformatics, and genetic techniques to prioritize potential drug targets from Mtb for structural analysis. Our core structural genomics pipeline works with the individual laboratories to produce diffraction quality crystals of targeted proteins, and structural analysis will be completed by the individual laboratories. We also have capabilities for functional analysis and the virtual ligand screening to identify novel inhibitors for target validation. Our overarching goals are to increase the knowledge of Mtb pathogenesis using the TB research community to drive structural genomics, particularly related to persistence, develop a central repository for TB research reagents, and discover chemical inhibitors of drug targets for future development of lead compounds.
Assuntos
Antituberculosos/farmacologia , Cristalografia , Desenho de Fármacos , Mycobacterium tuberculosis/efeitos dos fármacos , Arginina/metabolismo , Proteínas de Bactérias/antagonistas & inibidores , Proteínas de Bactérias/química , Avaliação Pré-Clínica de Medicamentos , Ferro/metabolismo , Malato Sintase/antagonistas & inibidores , Malato Sintase/química , Técnicas Analíticas Microfluídicas , Proteínas de Transporte de Monossacarídeos/antagonistas & inibidores , Proteínas de Transporte de Monossacarídeos/química , Mycobacterium tuberculosis/genética , Mycobacterium tuberculosis/metabolismo , Ácidos Micólicos/antagonistas & inibidores , Peptídeo Sintases/antagonistas & inibidores , Peptídeo Sintases/química , Difração de Raios XRESUMO
Protein expression screening methods are essential for proteomic scale characterization of gene and cDNA expression libraries. Screening methods are also important for the identification of highly expressed protein targets, for example, in quantities suitable for high-throughput screening and protein structural studies. To address these needs, we describe the implementation of several rapid, fluorescence-based protein expression screening strategies using Escherichia coli or E. coli-based in vitro transcription/translation (IVT) systems. In vitro expression screening is fast, convenient and, as we show, correlates well with in vivo expression. For screening, expressed proteins are labeled either as fusions with green fluorescent protein (GFP) or through translational incorporation of a fluorescent amino acid derivative, BODIPY-FL-Lysine. Fluorescence-based detection of GFP fusions or BODIPY-labeled proteins is considerably faster than other common expression screening methods, such as immunological detection of gels or dot blots. Furthermore, in vitro and in vivo screening used together yield a larger set of expressed proteins than either method alone. Specifically labeled proteins in cellular lysates are detected in one of three formats: a microplate using a fluorescence plate reader, a dot-blot using a fluorescence scanner or a microarray using a laser scanner. We have established a correlation among the various detection formats, which validates the use of protein microarrays for expression screening. Production of expressed proteins detected through screening can be scaled up either using IVT reactions or with in vivo expression systems in the absence of a fluorophore for subsequent characterization of protein function or interactions.
Assuntos
Expressão Gênica , Biossíntese de Proteínas , Proteômica/métodos , Compostos de Boro/química , DNA Complementar/genética , Escherichia coli/genética , Escherichia coli/metabolismo , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Humanos , Immunoblotting , Plasmídeos/genética , Reação em Cadeia da Polimerase , Polivinil/química , Análise Serial de Proteínas/métodos , Aminoacil-RNA de Transferência/química , Aminoacil-RNA de Transferência/metabolismo , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Reprodutibilidade dos Testes , Rodaminas/química , Espectrometria de FluorescênciaAssuntos
Toxinas Botulínicas/química , Toxinas Botulínicas/metabolismo , Proteínas de Membrana/metabolismo , Apoproteínas/química , Apoproteínas/metabolismo , Sítios de Ligação , Toxinas Botulínicas Tipo A , Elétrons , Proteínas de Membrana/química , Modelos Moleculares , Ligação Proteica , Estrutura Terciária de Proteína , Proteínas R-SNARE , Reprodutibilidade dos Testes , Software , TermodinâmicaRESUMO
The major human abasic endonuclease, Ape1, is an essential DNA repair enzyme that initiates the removal of apurinic/apyrimidinic sites from DNA, excises 3' replication-blocking moieties, and modulates the DNA binding activity of several transcriptional regulators. We have determined the X-ray structure of the full-length human Ape1 enzyme in two new crystal forms, one at neutral and one at acidic pH. The new structures are generally similar to the previously determined structure of a truncated Ape1 protein, but differ in the conformation of several loop regions and in spans of residues with weak electron density. While only one active-site metal ion is present in the structure determined at low pH, the structure determined from a crystal grown at the pH optimum of Ape1 nuclease activity, pH 7.5, has two metal ions bound 5 A apart in the active site. Enzyme kinetic data indicate that at least two metal-binding sites are functionally important, since Ca(2+) exhibits complex stimulatory and inhibitory effects on the Mg(2+)-dependent catalysis of Ape1, even though Ca(2+) itself does not serve as a cofactor. In conjunction, the structural and kinetic data suggest that Ape1 catalyzes hydrolysis of the DNA backbone through a two metal ion-mediated mechanism.
Assuntos
Cátions Bivalentes/metabolismo , Exodesoxirribonucleases/metabolismo , Metais/metabolismo , Sítios de Ligação , Cálcio/metabolismo , Catálise , Coenzimas/metabolismo , Cristalização , Cristalografia por Raios X , DNA/genética , DNA/metabolismo , Proteínas de Ligação a DNA/química , Proteínas de Ligação a DNA/metabolismo , Exodesoxirribonucleases/química , Humanos , Concentração de Íons de Hidrogênio , Hidrólise , Cinética , Magnésio/metabolismo , Modelos Moleculares , Movimento (Física) , Oxirredução , Ligação Proteica , Estrutura Terciária de Proteína , Relação Estrutura-AtividadeRESUMO
Apolipoprotein E (apoE) is an important lipid-transport protein in human plasma and brain. It has three common isoforms (apoE2, apoE3, and apoE4). ApoE is a major genetic risk factor in heart disease and in neurodegenerative disease, including Alzheimer's disease. The interaction of apoE with heparan sulfate proteoglycans plays an important role in lipoprotein remnant uptake and likely in atherogenesis and Alzheimer's disease. Here we report our studies of the interaction of the N-terminal domain of apoE4 (residues 1-191), which contains the major heparin-binding site, with an enzymatically prepared heparin oligosaccharide. Identified by its high affinity for the N-terminal domain of apoE4, this oligosaccharide was determined to be an octasaccharide of the structure DeltaUAp2S(1-->[4)-alpha-D-GlcNpS6S(1-->4)-alpha-L-IdoAp2S(1-->](3)4)-alpha-D-GlcNpS6S by nuclear magnetic resonance spectroscopy, capillary electrophoresis, and polyacrylamide gel electrophoresis. Kinetic analysis of the interaction between the N-terminal apoE4 fragment and immobilized heparin by surface plasmon resonance yielded a K(d) of 150 nM. A similar binding constant (K(d) = 140 nM) was observed for the interaction between immobilized N-terminal apoE4 and the octasaccharide. Isothermal titration calorimetry revealed a K(d) of 75 nM for the interaction of the N-terminal apoE fragment and the octasaccharide with a binding stoichiometry of approximately 1:1. Using previous studies and molecular modeling, we propose a binding site for this octasaccharide in a basic residue-rich region of helix 4 of the N-terminal fragment. From the X-ray crystal structure of the N-terminal apoE4, we predicted that binding of the octasaccharide at this site would result in a change in intrinsic fluorescence. This prediction was confirmed experimentally by an observed increase in fluorescence intensity with octasaccharide binding corresponding to a K(d) of approximately 1 microM.
Assuntos
Apolipoproteínas E/metabolismo , Heparina/metabolismo , Fragmentos de Peptídeos/metabolismo , Animais , Apolipoproteína E4 , Apolipoproteínas E/química , Calorimetria , Sequência de Carboidratos , Cristalografia por Raios X , Heparina/química , Cinética , Dados de Sequência Molecular , Oligossacarídeos/química , Oligossacarídeos/metabolismo , Fragmentos de Peptídeos/química , Espectrometria de Fluorescência , Ressonância de Plasmônio de Superfície , SuínosRESUMO
An amino-terminal fragment of human apolipoprotein E3 (residues 1-165) has been expressed and crystallized in three different crystal forms under similar crystallization conditions. One crystal form has nearly identical cell dimensions to the previously reported orthorhombic (P2(1)2(1)2(1)) crystal form of the amino-terminal 22 kDa fragment of apolipoprotein E (residues 1-191). A second orthorhombic crystal form (P2(1)2(1)2(1) with cell dimensions differing from the first form) and a trigonal (P3(1)21) crystal form were also characterized. The structures of the first orthorhombic and the trigonal form were determined by seleno-methionine multiwavelength anomalous dispersion, and the structure of the second orthorhombic form was determined by molecular replacement using the structure from the trigonal form as a search model. A combination of modern experimental and computational techniques provided high-quality electron-density maps, which revealed new features of the apolipoprotein E structure, including an unambiguously traced loop connecting helices 2 and 3 in the four-helix bundle and a number of multiconformation side chains. The three crystal forms contain a common intermolecular, antiparallel packing arrangement. The electrostatic complimentarity observed in this antiparallel packing resembles the interaction of apolipoprotein E with the monoclonal antibody 2E8 and the low density lipoprotein receptor. Superposition of the model structures from all three crystal forms reveals flexibility and pronounced kinks in helices near one end of the four-helix bundle. This mobility at one end of the molecule provides new insights into the structural changes in apolipoprotein E that occur with lipid association.
Assuntos
Apolipoproteínas E/química , Metabolismo dos Lipídeos , Animais , Apolipoproteínas/química , Cristalografia por Raios X , Elétrons , Gafanhotos , Humanos , Modelos Moleculares , Mariposas , Ligação Proteica , Conformação Proteica , Estrutura Secundária de Proteína , Estrutura Terciária de Proteína , Receptores de LDL/química , Receptores de LDL/metabolismoAssuntos
Antígenos CD1/fisiologia , Animais , Células Apresentadoras de Antígenos/imunologia , Células Apresentadoras de Antígenos/fisiologia , Antígenos CD1/química , Antígenos CD1/imunologia , Cristalografia por Raios X , Humanos , Modelos Moleculares , Conformação Proteica , Linfócitos T/imunologiaRESUMO
Three crystal forms of Naja naja naja phospholipase A2 were discovered through random crystallization screening, including two heretofore uncharacterized forms. The crystallization conditions for both of these novel crystal forms are Ca(2+)-free whereas previously reported conditions include Ca2+. One of the new crystal forms has a cubic lattice in the space group P2(1)3 (a = b = c = 69.24 A), the other has an orthorhombic lattice in the space group P2(1)2(1)2(1) (a = 67.22 A, b = 73.48 A, c = 87.52 A) and a previously characterized crystal belong to the tetragonal space group P4(3)2(1)2 (a = b = 88.6 A, c = 107.4 A). The structure from the cubic crystal form has been determined to 1.8 A and refined to an R-factor of 17% while the structure from the orthorhombic form has been determined to 2.65 A and has been refined to an R-factor of 21%. The determination of the cubic structure extends the resolution to which structures of this molecule have been determined from 2.3 A to 1.8 A. The two newly determined structures, in combination with the previously determined structure, generate an informative structural ensemble from which structural changes due to Ca2+, which is required for catalysis, and the effect of crystal contacts on side-chain conformations and oligomeric association can be inferred. Both of the newly determined structures reveal a trimeric oligomer as observed in the tetragonal structure; this appears to be a unique feature of the Naja naja naja enzyme.
Assuntos
Cálcio/deficiência , Venenos Elapídicos/química , Fosfolipases A/química , Conformação Proteica , Animais , Cálcio/química , Cristalografia por Raios X , Modelos Moleculares , Fosfolipases A2 , PolímerosRESUMO
CD1 represents a third lineage of antigen-presenting molecules that are distantly related to major histocompatibility complex (MHC) molecules in the immune system. The crystal structure of mouse CD1d1, corresponding to human CD1d, at 2.8 resolution shows that CD1 adopts an MHC fold that is more closely related to that of MHC class I than to that of MHC class II. The binding groove, although significantly narrower, is substantially larger because of increased depth and it has only two major pockets that are almost completely hydrophobic. The extreme hydrophobicity and shape of the binding site are consistent with observations that human CD1b and CD1c can present mycobacterial cell wall antigens, such as mycolic acid and lipoarabinomannans. However, mouse CD1d1 can present very hydrophobic peptides, but must do so in a very different way from MHC class Ia and class II molecules.
Assuntos
Apresentação de Antígeno , Antígenos CD1/química , Conformação Proteica , Dobramento de Proteína , Animais , Antígenos CD1/imunologia , Antígenos CD1/metabolismo , Sítios de Ligação , Cristalização , Cristalografia por Raios X , Glicolipídeos/química , Glicolipídeos/imunologia , Glicolipídeos/metabolismo , Antígenos de Histocompatibilidade Classe I/química , Antígenos de Histocompatibilidade Classe II/química , Humanos , Ligação de Hidrogênio , Ligantes , Metabolismo dos Lipídeos , Lipídeos/química , Lipídeos/imunologia , Camundongos , Modelos Moleculares , Estrutura Secundária de Proteína , Estrutura Terciária de Proteína , Subpopulações de Linfócitos T/imunologiaRESUMO
Transferred NOE experiments have been carried out on cobra venom (Naja naja naja) phospholipase A2 (PLA2) with substrate analogues which serve as potent inhibitors. 1-(Hexylthio)-2-(hexanoylamino)-1,2-dideoxy-sn-glycero-3-pho sphoethanolamine (PE) and the corresponding phosphocholine analogue (PC) are water-soluble, short-chain, nonhydrolyzable substrate analogues which bind tightly to the enzyme. Because they are small compounds and monomeric in solution, NOEs develop inefficiently in the absence of enzyme. Thus, the PLA2/inhibitor system is ideal for analyzing transferred NOEs. The experiments are carried out under conditions that are optimal for catalysis, pH 7.5 in the presence of 2 mM CaCl2. The data show the inhibitor conformation in the catalytic site of cobra PLA2 in solution. The effect of the thioether in the sn-1 chain on the chemical shift dispersion of the methylene protons allowed for chain-specific assignments and detailed conformational analysis. Both inhibitors adopt a PLA2-bound conformation in which the end of the sn-2 chain is within 5 A of the alpha-methylene of the sn-1 chain. In addition, intermolecular contact points between the inhibitor and the enzyme were identified by NOEs.