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1.
Proc Natl Acad Sci U S A ; 120(37): e2303937120, 2023 09 12.
Artigo em Inglês | MEDLINE | ID: mdl-37669369

RESUMO

While some agricultural landscapes can support wildlife in the short term, it is uncertain how well they can truly sustain wildlife populations. To compare population trends in different production systems, we sampled birds along 48 transects in mature forests, diversified farms, and intensive farms across Costa Rica from 2000 to 2017. To assess how land use influenced population trends in the 349 resident and 80 migratory species with sufficient data, we developed population models. We found, first, that 23% of species were stable in all three land use types, with the rest almost evenly split between increasing and decreasing populations. Second, in forest habitats, a slightly higher fraction was declining: 62% of the 164 species undergoing long-term population changes; nearly half of these declines occurred in forest-affiliated invertivores. Third, in diversified farms, 49% of the 230 species with population changes were declining, with 60% of these declines occurring in agriculture-affiliated species. In contrast, 51% of the species with population changes on diversified farms showed increases, primarily in forest-affiliated invertivores and frugivores. In intensive farms, 153 species showed population changes, also with similar proportions of species increasing (50%) and decreasing (50%). Declines were concentrated in agriculture-affiliated invertivores and forest-affiliated frugivores; increases occurred in many large, omnivorous species. Our findings paint a complex picture but clearly indicate that diversified farming helps sustain populations of diverse, forest-affiliated species. Despite not fully offsetting losses in forest habitats, diversified farming practices help sustain wildlife in a critical time, before possible transformation to nature-positive policies and practices.


Assuntos
Agricultura , Florestas , Animais , Fazendas , Animais Selvagens , Aves
2.
FASEB J ; 36(7): e22378, 2022 07.
Artigo em Inglês | MEDLINE | ID: mdl-35639414

RESUMO

Structural discovery of guanine nucleotide exchange factor (GEF) protein complexes is likely to become increasingly relevant with the development of new therapeutics targeting small GTPases and development of new classes of small molecules that inhibit protein-protein interactions. Syx (also known as PLEKHG5 in humans) is a RhoA GEF implicated in the pathology of glioblastoma (GBM). Here we investigated protein expression and purification of ten different human Syx constructs and performed biophysical characterizations and computational studies that provide insights into why expression of this protein was previously intractable. We show that human Syx can be expressed and isolated and Syx is folded as observed by circular dichroism (CD) spectroscopy and actively binds to RhoA as determined by co-elution during size exclusion chromatography (SEC). This characterization may provide critical insights into the expression and purification of other recalcitrant members of the large class of oncogenic-Diffuse B-cell lymphoma (Dbl) homology GEF proteins. In addition, we performed detailed homology modeling and molecular dynamics simulations on the surface of a physiologically realistic membrane. These simulations reveal novel insights into GEF activity and allosteric modulation by the plekstrin homology (PH) domain. These newly revealed interactions between the GEF PH domain and the membrane embedded region of RhoA support previously unexplained experimental findings regarding the allosteric effects of the PH domain from numerous activity studies of Dbl homology GEF proteins. This work establishes new hypotheses for structural interactivity and allosteric signal modulation in Dbl homology RhoGEFs.


Assuntos
Glioblastoma , Fatores de Troca de Nucleotídeo Guanina Rho , Glioblastoma/genética , Fatores de Troca do Nucleotídeo Guanina , Humanos , Proteínas , Fatores de Troca de Nucleotídeo Guanina Rho/genética
3.
Protein Expr Purif ; 185: 105890, 2021 09.
Artigo em Inglês | MEDLINE | ID: mdl-33971243

RESUMO

Human G-protein coupled receptor kinase 6 (GRK6) belongs to the GRK4 kinase subfamily of the G protein-coupled receptor kinase family which comprises of GRK1, GRK2, and GRK4. These kinases phosphorylate ligand-activated G-protein coupled receptors (GPCRs), driving heterotrimeric G protein coupling, desensitization of GPCR, and ß-arrestin recruitment. This reaction series mediates cellular signal pathways for cell survival, proliferation, migration and chemotaxis. GRK6 is a kinase target in multiple myeloma since it is highly expressed in myeloma cells compared to epithelial cells and has a significant role in mediating the chemotactic responses of T and B-lymphocytes. To support structure-based drug design, we describe three human GRK6 constructs, GRK6, GRK6His/EK, and GRK6His/TEV, designed for protein expression in Spodoptera frugiperda Sf9 insect cells. The first construct did not contain any purification tag whereas the other two constructs contained the His10 affinity tag, which increased purification yields. We report here that all three constructs of GRK6 were overexpressed in Sf9 insect cells and purified to homogeneity at levels that were suitable for co-crystallization of GRK6 with potential inhibitors. The yields of purified GRK6, GRK6His/EK, and GRK6His/TEV were 0.3 mg, 0.8 mg and 0.7 mg per liter of cell culture, respectively. In addition, we have shown that GRK6His/TEV with the His10 tag removed was highly homogeneous and monodisperse as observed by dynamic light scattering measurement and actively folded as exhibited by circular dichroism spectroscopy. The described methods will support the structure-based development of additional therapeutics against multiple myeloma.


Assuntos
Quinases de Receptores Acoplados a Proteína G/isolamento & purificação , Proteínas de Neoplasias/isolamento & purificação , Proteínas Recombinantes de Fusão/isolamento & purificação , Animais , Antineoplásicos/síntese química , Baculoviridae/genética , Baculoviridae/metabolismo , Cromatografia/métodos , Clonagem Molecular , Desenho de Fármacos , Quinases de Receptores Acoplados a Proteína G/química , Quinases de Receptores Acoplados a Proteína G/genética , Quinases de Receptores Acoplados a Proteína G/metabolismo , Expressão Gênica , Vetores Genéticos/química , Vetores Genéticos/metabolismo , Humanos , Mieloma Múltiplo/tratamento farmacológico , Mieloma Múltiplo/enzimologia , Mieloma Múltiplo/genética , Proteínas de Neoplasias/química , Proteínas de Neoplasias/genética , Proteínas de Neoplasias/metabolismo , Conformação Proteica , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Células Sf9 , Spodoptera
4.
BMC Biol ; 16(1): 59, 2018 05 31.
Artigo em Inglês | MEDLINE | ID: mdl-29848358

RESUMO

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.


Assuntos
Antibacterianos/química , Proteínas de Bactérias/química , Ceftriaxona/química , Cristalografia por Raios X/métodos , Mycobacterium tuberculosis/enzimologia , beta-Lactamases/química , Proteínas de Bactérias/genética , Biocatálise , Resistência às Cefalosporinas/genética , Cinética , Lasers , Modelos Moleculares , Fatores de Tempo , beta-Lactamases/genética
5.
Biochemistry ; 53(12): 1958-70, 2014 Apr 01.
Artigo em Inglês | MEDLINE | ID: mdl-24593131

RESUMO

The capA gene (FTT0807) from Francisella tularensis subsp. tularensis SCHU S4 encodes a 44.4 kDa integral membrane protein composed of 403 amino acid residues that is part of an apparent operon that encodes at least two other membrane proteins, CapB, and CapC, which together play a critical role in the virulence and pathogenesis of this bacterium. The capA gene was overexpressed in Escherichia coli as a C-terminal His6-tagged fusion with a folding reporter green fluorescent protein (frGFP). Purification procedures using several detergents were developed for the fluorescing and membrane-bound product, yielding approximately 30 mg of pure protein per liter of bacterial culture. Dynamic light scattering indicated that CapA-frGFP was highly monodisperse, with a size that was dependent upon both the concentration and choice of detergent. Circular dichroism showed that CapA-frGFP was stable over the range of 3-9 for the pH, with approximately half of the protein having well-defined α-helical and ß-sheet secondary structure. The addition of either sodium chloride or calcium chloride at concentrations producing ionic strengths above 0.1 M resulted in a small increase of the α-helical content and a corresponding decrease in the random-coil content. Secondary-structure predictions on the basis of the analysis of the sequence indicate that the CapA membrane protein has two transmembrane helices with a substantial hydrophilic domain. The hydrophilic domain is predicted to contain a long disordered region of 50-60 residues, suggesting that the increase of α-helical content at high ionic strength could arise because of electrostatic interactions involving the disordered region. CapA is shown to be an inner-membrane protein and is predicted to play a key cellular role in the assembly of polysaccharides.


Assuntos
Proteínas de Bactérias/isolamento & purificação , Proteínas de Bactérias/fisiologia , Francisella tularensis/química , Francisella tularensis/fisiologia , Proteínas de Choque Térmico/isolamento & purificação , Proteínas de Choque Térmico/fisiologia , Sequência de Aminoácidos , Proteínas de Bactérias/química , Fenômenos Biofísicos/fisiologia , Proteínas de Choque Térmico/química , Dados de Sequência Molecular , Valor Preditivo dos Testes
6.
J Am Chem Soc ; 136(23): 8283-95, 2014 Jun 11.
Artigo em Inglês | MEDLINE | ID: mdl-24871902

RESUMO

Proteins have evolved to carry out nearly all the work required of living organisms within complex inter- and intracellular environments. However, systematically investigating the range of interactions experienced by a protein that influence its function remains challenging. DNA nanostructures are emerging as a convenient method to arrange a broad range of guest molecules. However, flexible methods are needed for arranging proteins in more biologically relevant 3D geometries under mild conditions that preserve protein function. Here we demonstrate how peptide nucleic acid (PNA) can be used to control the assembly of cytochrome c (12.5 kDa, pI 10.5) and azurin (13.9 kDa, pI 5.7) proteins into separate 3D DNA nanocages, in a process that maintains protein function. Toehold-mediated DNA strand displacement is introduced as a method to purify PNA-protein conjugates. The PNA-proteins were assembled within 2 min at room temperature and within 4 min at 11 °C, and hybridize with even greater efficiency than PNA conjugated to a short peptide. Gel electrophoresis and steady state and time-resolved fluorescence spectroscopy were used to investigate the effect of protein surface charge on its interaction with the negatively charged DNA nanocage. These data were used to generate a model of the DNA-PNA-protein complexes that show the negatively charged azurin protein repelled away from the DNA nanocage while the positively charged cytochrome c protein remains within and closely interacts with the DNA nanocage. When conjugated to PNA and incorporated into the DNA nanocage, the cytochrome c secondary structure and catalytic activity were maintained, and its redox potential was reduced modestly by 20 mV possibly due to neutralization of some positive surface charges. This work demonstrates a flexible new approach for using 3D nucleic acid (PNA-DNA) nanostructures to control the assembly of functional proteins, and facilitates further investigation of protein interactions as well as engineer more elaborate 3D protein complexes.


Assuntos
Azurina/química , Citocromos c/química , DNA/química , Nanoestruturas/química , Ácidos Nucleicos Peptídicos/química , Temperatura , Cromatografia em Gel , Modelos Moleculares , Espectrometria de Fluorescência
7.
Sci Rep ; 12(1): 11824, 2022 07 12.
Artigo em Inglês | MEDLINE | ID: mdl-35821229

RESUMO

Particulate Guanylyl Cyclase Receptor A (pGC-A) is a natriuretic peptide membrane receptor, playing a vital role in controlling cardiovascular, renal, and endocrine functions. The extracellular domain interacts with natriuretic peptides and triggers the intracellular guanylyl cyclase domain to convert GTP to cGMP. To effectively develop methods to regulate pGC-A, structural information on the full-length form is needed. However, structural data on the transmembrane and intracellular domains are lacking. This work presents expression and optimization using baculovirus, along with the first purification of functional full-length human pGC-A. In vitro assays revealed the pGC-A tetramer was functional in detergent micelle solution. Based on our purification results and previous findings that dimer formation is required for functionality, we propose a tetramer complex model with two functional subunits. Previous research suggested pGC-A signal transduction is an ATP-dependent, two-step mechanism. Our results show the binding ligand also moderately activates pGC-A, and ATP is not crucial for activation of guanylyl cyclase. Furthermore, crystallization of full-length pGC-A was achieved, toward determination of its structure. Needle-shaped crystals with 3 Å diffraction were observed by serial crystallography. This work paves the road for determination of the full-length pGC-A structure and provides new information on the signal transduction mechanism.


Assuntos
Guanilato Ciclase , Receptores do Fator Natriurético Atrial , Trifosfato de Adenosina/metabolismo , Cristalografia , Poeira , Guanilato Ciclase/metabolismo , Humanos , Receptores do Fator Natriurético Atrial/metabolismo , Receptores Acoplados a Guanilato Ciclase
8.
PLoS One ; 17(8): e0267370, 2022.
Artigo em Inglês | MEDLINE | ID: mdl-35913965

RESUMO

Francisella tularensis is an extremely infectious pathogen and a category A bioterrorism agent. It causes the highly contagious zoonosis, Tularemia. Currently, FDA approved vaccines against tularemia are unavailable. F. tularensis outer membrane protein A (FopA) is a well-studied virulence determinant and protective antigen against tularemia. It is a major outer membrane protein (Omp) of F. tularensis. However, FopA-based therapeutic intervention is hindered due to lack of complete structural information for membrane localized mature FopA. In our study, we established recombinant expression, monodisperse purification, crystallization and X-ray diffraction (~6.5 Å) of membrane localized mature FopA. Further, we performed bioinformatics and biophysical experiments to unveil its structural organization in the outer membrane. FopA consists of 393 amino acids and has less than 40% sequence identity to known bacterial Omps. Using comprehensive sequence alignments and structure predictions together with existing partial structural information, we propose a two-domain organization for FopA. Circular dichroism spectroscopy and heat modifiability assay confirmed FopA has a ß-barrel domain consistent with alphafold2's prediction of an eight stranded ß-barrel at the N-terminus. Small angle X-ray scattering (SAXS) and native-polyacrylamide gel electrophoresis revealed FopA purified in detergent micelles is predominantly dimeric. Molecular density derived from SAXS at 31 Å shows putative dimeric N-terminal ß-barrels surrounded by detergent corona and connected to C-terminal domains via flexible linker. Disorder analysis predicts N- and C-terminal domains are interspersed by a long intrinsically disordered region and alphafold2 predicts this region to be largely unstructured. Taken together, we propose a dimeric, two-domain organization of FopA in the outer membrane: the N-terminal ß-barrel is membrane embedded, provides dimerization interface and tethers to membrane extrinsic C-terminal domain via long flexible linker. Structure determination of membrane localized mature FopA is essential to understand its role in pathogenesis and develop anti-tularemia therapeutics. Our results pave the way towards it.


Assuntos
Francisella tularensis , Tularemia , Detergentes , Humanos , Espalhamento a Baixo Ângulo , Tularemia/microbiologia , Difração de Raios X
9.
J Appl Crystallogr ; 55(Pt 1): 1-13, 2022 Feb 01.
Artigo em Inglês | MEDLINE | ID: mdl-35153640

RESUMO

Serial femtosecond crystallography (SFX) is a powerful technique that exploits X-ray free-electron lasers to determine the structure of macro-molecules at room temperature. Despite the impressive exposition of structural details with this novel crystallographic approach, the methods currently available to introduce crystals into the path of the X-ray beam sometimes exhibit serious drawbacks. Samples requiring liquid injection of crystal slurries consume large quantities of crystals (at times up to a gram of protein per data set), may not be compatible with vacuum configurations on beamlines or provide a high background due to additional sheathing liquids present during the injection. Proposed and characterized here is the use of an immiscible inert oil phase to supplement the flow of sample in a hybrid microfluidic 3D-printed co-flow device. Co-flow generation is reported with sample and oil phases flowing in parallel, resulting in stable injection conditions for two different resin materials experimentally. A numerical model is presented that adequately predicts these flow-rate conditions. The co-flow generating devices reduce crystal clogging effects, have the potential to conserve protein crystal samples up to 95% and will allow degradation-free light-induced time-resolved SFX.

10.
Protein Expr Purif ; 80(2): 157-68, 2011 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-21878393

RESUMO

Membrane proteins compose more than 30% of all proteins in the living cell. However, many membrane proteins have low abundance in the cell and cannot be isolated from natural sources in concentrations suitable for structure analysis. The overexpression, reconstitution, and stabilization of membrane proteins are complex and remain a formidable challenge in membrane protein characterization. Here we describe a novel, in vitro folding procedure for a cation-selective channel protein, the outer envelope membrane protein 16 (OEP16) of pea chloroplast, overexpressed in Escherichia coli in the form of inclusion bodies. The protein is purified and then folded with detergent on a Ni-NTA affinity column. Final concentrations of reconstituted OEP16 of up to 24 mg/ml have been achieved, which provides samples that are sufficient for structural studies by NMR and crystallography. Reconstitution of OEP16 in detergent micelles was monitored by circular dichroism, fluorescence, and NMR spectroscopy. Tryptophan fluorescence spectra of heterologous expressed OEP16 in micelles are similar to spectra of functionally active OEP16 in liposomes, which indicates folding of the membrane protein in detergent micelles. CD spectroscopy studies demonstrate a folded protein consisting primarily of α-helices. ¹5N-HSQC NMR spectra also provide evidence for a folded protein. We present here a convenient, effective and quantitative method to screen large numbers of conditions for optimal protein stability by using microdialysis chambers in combination with fluorescence spectroscopy. Recent collection of multidimensional NMR data at 500, 600 and 800 MHz demonstrated that the protein is suitable for structure determination by NMR and stable for weeks during data collection.


Assuntos
Sistemas de Transporte de Aminoácidos/química , Proteínas de Membrana/química , Pisum sativum/química , Proteínas de Plantas/química , Dobramento de Proteína , Sistemas de Transporte de Aminoácidos/genética , Sistemas de Transporte de Aminoácidos/isolamento & purificação , Cloroplastos/química , Cloroplastos/genética , Cromatografia de Afinidade , Dicroísmo Circular , Detergentes/química , Escherichia coli/química , Escherichia coli/genética , Genes de Plantas , Corpos de Inclusão/química , Membranas Intracelulares/química , Lipossomos/química , Espectroscopia de Ressonância Magnética , Proteínas de Membrana/genética , Proteínas de Membrana/isolamento & purificação , Micelas , Microdiálise/métodos , Pisum sativum/genética , Proteínas de Plantas/genética , Proteínas de Plantas/isolamento & purificação , Estabilidade Proteica , Estrutura Secundária de Proteína , Proteínas Recombinantes/química , Proteínas Recombinantes/isolamento & purificação , Dodecilsulfato de Sódio/química , Solubilidade , Triptofano/química , Ultrafiltração/métodos
11.
PLoS One ; 16(7): e0249164, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34260600

RESUMO

In the United States non-alcoholic fatty liver disease (NAFLD) is the most common form of chronic liver disease, affecting an estimated 80 to 100 million people. It occurs in every age group, but predominantly in people with risk factors such as obesity and type 2 diabetes. NAFLD is marked by fat accumulation in the liver leading to liver inflammation, which may lead to scarring and irreversible damage progressing to cirrhosis and liver failure. In animal models, genetic ablation of the protein G0S2 leads to alleviation of liver damage and insulin resistance in high fat diets. The research presented in this paper aims to aid in rational based drug design for the treatment of NAFLD by providing a pathway for a solution state NMR structure of G0S2. Here we describe the expression of G0S2 in an E. coli system from two different constructs, both of which are confirmed to be functionally active based on the ability to inhibit the activity of Adipose Triglyceride Lipase. In one of the constructs, preliminary NMR spectroscopy measurements show dominant alpha-helical characteristics as well as resonance assignments on the N-terminus of G0S2, allowing for further NMR work with this protein. Additionally, the characterization of G0S2 oligomers are outlined for both constructs, suggesting that G0S2 may defensively exist in a multimeric state to protect and potentially stabilize the small 104 amino acid protein within the cell. This information presented on the structure of G0S2 will further guide future development in the therapy for NAFLD.


Assuntos
Proteínas de Ciclo Celular/química , Proteínas de Ciclo Celular/metabolismo , Espectroscopia de Ressonância Magnética , Hepatopatia Gordurosa não Alcoólica/enzimologia , Animais , Humanos
12.
Structure ; 28(5): 540-547.e3, 2020 05 05.
Artigo em Inglês | MEDLINE | ID: mdl-32142641

RESUMO

Francisella tularensis is the causative agent for the potentially fatal disease tularemia. The lipoprotein Flpp3 has been identified as a virulence determinant of tularemia with no sequence homology outside the Francisella genus. We report a room temperature structure of Flpp3 determined by serial femtosecond crystallography that exists in a significantly different conformation than previously described by the NMR-determined structure. Furthermore, we investigated the conformational space and energy barriers between these two structures by molecular dynamics umbrella sampling and identified three low-energy intermediate states, transitions between which readily occur at room temperature. We have also begun to investigate organic compounds in silico that may act as inhibitors to Flpp3. This work paves the road to developing targeted therapeutics against tularemia and aides in our understanding of the disease mechanisms of tularemia.


Assuntos
Antibacterianos/química , Francisella tularensis , Lipoproteínas/química , Antibacterianos/farmacologia , Cristalografia por Raios X/métodos , Bases de Dados de Produtos Farmacêuticos , Avaliação Pré-Clínica de Medicamentos/métodos , Francisella tularensis/química , Francisella tularensis/patogenicidade , Humanos , Interações Hidrofóbicas e Hidrofílicas , Lasers , Lipoproteínas/antagonistas & inibidores , Lipoproteínas/genética , Simulação de Dinâmica Molecular , Terapia de Alvo Molecular , Ressonância Magnética Nuclear Biomolecular , Conformação Proteica , Tularemia/tratamento farmacológico , Fatores de Virulência/química
13.
IUCrJ ; 4(Pt 4): 439-454, 2017 Jul 01.
Artigo em Inglês | MEDLINE | ID: mdl-28875031

RESUMO

Crystal structure determination of biological macromolecules using the novel technique of serial femtosecond crystallography (SFX) is severely limited by the scarcity of X-ray free-electron laser (XFEL) sources. However, recent and future upgrades render microfocus beamlines at synchrotron-radiation sources suitable for room-temperature serial crystallography data collection also. Owing to the longer exposure times that are needed at synchrotrons, serial data collection is termed serial millisecond crystallography (SMX). As a result, the number of SMX experiments is growing rapidly, with a dozen experiments reported so far. Here, the first high-viscosity injector-based SMX experiments carried out at a US synchrotron source, the Advanced Photon Source (APS), are reported. Microcrystals (5-20 µm) of a wide variety of proteins, including lysozyme, thaumatin, phycocyanin, the human A2A adenosine receptor (A2AAR), the soluble fragment of the membrane lipoprotein Flpp3 and proteinase K, were screened. Crystals suspended in lipidic cubic phase (LCP) or a high-molecular-weight poly(ethylene oxide) (PEO; molecular weight 8 000 000) were delivered to the beam using a high-viscosity injector. In-house data-reduction (hit-finding) software developed at APS as well as the SFX data-reduction and analysis software suites Cheetah and CrystFEL enabled efficient on-site SMX data monitoring, reduction and processing. Complete data sets were collected for A2AAR, phycocyanin, Flpp3, proteinase K and lysozyme, and the structures of A2AAR, phycocyanin, proteinase K and lysozyme were determined at 3.2, 3.1, 2.65 and 2.05 Šresolution, respectively. The data demonstrate the feasibility of serial millisecond crystallography from 5-20 µm crystals using a high-viscosity injector at APS. The resolution of the crystal structures obtained in this study was dictated by the current flux density and crystal size, but upcoming developments in beamline optics and the planned APS-U upgrade will increase the intensity by two orders of magnitude. These developments will enable structure determination from smaller and/or weakly diffracting microcrystals.

14.
Struct Dyn ; 4(4): 044003, 2017 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-28083542

RESUMO

Mix-and-inject serial crystallography (MISC) is a technique designed to image enzyme catalyzed reactions in which small protein crystals are mixed with a substrate just prior to being probed by an X-ray pulse. This approach offers several advantages over flow cell studies. It provides (i) room temperature structures at near atomic resolution, (ii) time resolution ranging from microseconds to seconds, and (iii) convenient reaction initiation. It outruns radiation damage by using femtosecond X-ray pulses allowing damage and chemistry to be separated. Here, we demonstrate that MISC is feasible at an X-ray free electron laser by studying the reaction of M. tuberculosis ß-lactamase microcrystals with ceftriaxone antibiotic solution. Electron density maps of the apo-ß-lactamase and of the ceftriaxone bound form were obtained at 2.8 Å and 2.4 Å resolution, respectively. These results pave the way to study cyclic and non-cyclic reactions and represent a new field of time-resolved structural dynamics for numerous substrate-triggered biological reactions.

15.
J Virol Methods ; 236: 237-244, 2016 10.
Artigo em Inglês | MEDLINE | ID: mdl-27465218

RESUMO

Inherent in the study of viruses is the risk of pathogenic exposure, which necessitates appropriate levels of biosafety containment. Unfortunately, this also limits the availability of useful research instruments that are located at facilities not equipped to handle infectious pathogens. Abrogation of viral infectivity can be accomplished without severely disrupting the physical structure of the virus particle. Virus samples that are verifiably intact but not infectious may be enabled for study at research facilities where they would otherwise not be allowed. Inactivated viruses are also used in the development of vaccines, where immunogenicity is sought in the absence of active infection. We demonstrate the inactivation of Sindbis alphavirus particles in solution, as well as in crystallized form. Inactivation was accomplished by two different approaches: crosslinking of proteins by glutaraldehyde treatment, and crosslinking of nucleic acids by UV irradiation. Biophysical characterization methods, including dynamic light scattering and transmission electron microscopy, were used to demonstrate that the glutaraldehyde and UV inactivated Sindbis virus particles remain intact structurally. SDS-PAGE was also used to show evidence of the protein crosslinking that was expected with glutaraldehyde treatment, but also observed with UV irradiation.


Assuntos
Cristalização , Sindbis virus/química , Vírion/química , Inativação de Vírus , Reagentes de Ligações Cruzadas/metabolismo , Desinfetantes/metabolismo , Difusão Dinâmica da Luz , Eletroforese em Gel de Poliacrilamida , Glutaral/metabolismo , Microscopia Eletrônica de Transmissão , Sindbis virus/fisiologia , Raios Ultravioleta
16.
Sci Rep ; 6: 35279, 2016 10 19.
Artigo em Inglês | MEDLINE | ID: mdl-27756898

RESUMO

Phytochromes are a family of photoreceptors that control light responses of plants, fungi and bacteria. A sequence of structural changes, which is not yet fully understood, leads to activation of an output domain. Time-resolved serial femtosecond crystallography (SFX) can potentially shine light on these conformational changes. Here we report the room temperature crystal structure of the chromophore-binding domains of the Deinococcus radiodurans phytochrome at 2.1 Å resolution. The structure was obtained by serial femtosecond X-ray crystallography from microcrystals at an X-ray free electron laser. We find overall good agreement compared to a crystal structure at 1.35 Å resolution derived from conventional crystallography at cryogenic temperatures, which we also report here. The thioether linkage between chromophore and protein is subject to positional ambiguity at the synchrotron, but is fully resolved with SFX. The study paves the way for time-resolved structural investigations of the phytochrome photocycle with time-resolved SFX.


Assuntos
Cristalografia por Raios X , Deinococcus/química , Fitocromo/química , Conformação Proteica , Cristalização , Temperatura
17.
Behav Ecol ; 26(3): 839-842, 2015.
Artigo em Inglês | MEDLINE | ID: mdl-26023277

RESUMO

Many animals communicate through acoustic signaling, and "acoustic space" may be viewed as a limited resource that organisms compete for. If acoustic signals overlap, the information in them is masked, so there should be selection toward strategies that reduce signal overlap. The extent to which animals are able to partition acoustic space in acoustically diverse habitats such as tropical forests is poorly known. Here, we demonstrate that a single cicada species plays a major role in the frequency and timing of acoustic communication in a neotropical wet forest bird community. Using an automated acoustic monitor, we found that cicadas vary the timing of their signals throughout the day and that the frequency range and timing of bird vocalizations closely track these signals. Birds significantly avoid temporal overlap with cicadas by reducing and often shutting down vocalizations at the onset of cicada signals that utilize the same frequency range. When birds do vocalize at the same time as cicadas, the vocalizations primarily occur at nonoverlapping frequencies with cicada signals. Our results greatly improve our understanding of the community dynamics of acoustic signaling and reveal how patterns in biotic noise shape the frequency and timing of bird vocalizations in tropical forests.

18.
Structure ; 23(6): 1116-22, 2015 Jun 02.
Artigo em Inglês | MEDLINE | ID: mdl-26004443

RESUMO

Tularemia is a potentially fatal bacterial infection caused by Francisella tularensis, and is endemic to North America and many parts of northern Europe and Asia. The outer membrane lipoprotein, Flpp3, has been identified as a virulence determinant as well as a potential subunit template for vaccine development. Here we present the first structure for the soluble domain of Flpp3 from the highly infectious Type A SCHU S4 strain, derived through high-resolution solution nuclear magnetic resonance (NMR) spectroscopy; the first structure of a lipoprotein from the genus Francisella. The Flpp3 structure demonstrates a globular protein with an electrostatically polarized surface containing an internal cavity-a putative binding site based on the structurally homologous Bet v1 protein family of allergens. NMR-based relaxation studies suggest loop regions that potentially modulate access to the internal cavity. The Flpp3 structure may add to the understanding of F. tularensis virulence and contribute to the development of effective vaccines.


Assuntos
Antígenos de Plantas/química , Francisella tularensis/química , Modelos Moleculares , Homologia Estrutural de Proteína , Fatores de Virulência/química , Biofísica , Western Blotting , Dicroísmo Circular , Eletroforese em Gel de Poliacrilamida , Simulação de Dinâmica Molecular , Ressonância Magnética Nuclear Biomolecular , Ligação Proteica , Conformação Proteica , Eletricidade Estática , Fatores de Virulência/isolamento & purificação
19.
IUCrJ ; 2(Pt 4): 421-30, 2015 Jul 01.
Artigo em Inglês | MEDLINE | ID: mdl-26177184

RESUMO

Serial femtosecond crystallography (SFX) has opened a new era in crystallo-graphy by permitting nearly damage-free, room-temperature structure determination of challenging proteins such as membrane proteins. In SFX, femtosecond X-ray free-electron laser pulses produce diffraction snapshots from nanocrystals and microcrystals delivered in a liquid jet, which leads to high protein consumption. A slow-moving stream of agarose has been developed as a new crystal delivery medium for SFX. It has low background scattering, is compatible with both soluble and membrane proteins, and can deliver the protein crystals at a wide range of temperatures down to 4°C. Using this crystal-laden agarose stream, the structure of a multi-subunit complex, phycocyanin, was solved to 2.5 Šresolution using 300 µg of microcrystals embedded into the agarose medium post-crystallization. The agarose delivery method reduces protein consumption by at least 100-fold and has the potential to be used for a diverse population of proteins, including membrane protein complexes.

20.
PLoS One ; 8(10): e78116, 2013.
Artigo em Inglês | MEDLINE | ID: mdl-24205117

RESUMO

Solving high-resolution structures for membrane proteins continues to be a daunting challenge in the structural biology community. In this study we report our high-resolution NMR results for a transmembrane protein, outer envelope protein of molar mass 16 kDa (OEP16), an amino acid transporter from the outer membrane of chloroplasts. Three-dimensional, high-resolution NMR experiments on the (13)C, (15)N, (2)H-triply-labeled protein were used to assign protein backbone resonances and to obtain secondary structure information. The results yield over 95% assignment of N, HN, CO, Cα, and Cß chemical shifts, which is essential for obtaining a high resolution structure from NMR data. Chemical shift analysis from the assignment data reveals experimental evidence for the first time on the location of the secondary structure elements on a per residue basis. In addition T 1Z and T2 relaxation experiments were performed in order to better understand the protein dynamics. Arginine titration experiments yield an insight into the amino acid residues responsible for protein transporter function. The results provide the necessary basis for high-resolution structural determination of this important plant membrane protein.


Assuntos
Sistemas de Transporte de Aminoácidos/química , Cloroplastos/metabolismo , Espectroscopia de Ressonância Magnética/métodos , Proteínas de Membrana/química , Proteínas de Plantas/química , Estrutura Secundária de Proteína
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