RESUMO
This review compares and discusses conventional versus miniaturized specimen preparation methods for transmission electron microscopy (TEM). The progress brought by direct electron detector cameras, software developments and automation have transformed transmission cryo-electron microscopy (cryo-EM) and made it an invaluable high-resolution structural analysis tool. In contrast, EM specimen preparation has seen very little progress in the last decades and is now one of the main bottlenecks in cryo-EM. Here, we discuss the challenges faced by specimen preparation for single particle EM, highlight current developments, and show the opportunities resulting from the advanced miniaturized and microfluidic sample grid preparation methods described, such as visual proteomics and time-resolved cryo-EM studies.
Assuntos
Microscopia Crioeletrônica/métodos , Imageamento Tridimensional/métodos , Microscopia Eletrônica de Transmissão/métodos , Proteínas/ultraestrutura , Proteômica/métodos , Humanos , Microfluídica/métodos , Manejo de EspécimesRESUMO
Two distinct dimers are formed during the initial steps of actin polymerization. The first one, referred to as the 'lower dimer' (LD) was discovered many years ago by means of chemical crosslinking. Owing to its transient nature, a biological relevance had long been precluded when, using LD-specific antibodies, we detected LD-like contacts in actin assemblies that are associated with the endolysosomal compartment in a number of different cell lines. Moreover, immunofluorescence showed the presence of LD-related structures at the cell periphery of migrating fibroblasts, in the nucleus, and in association with the centrosome of interphase cells. Here, we explore contributions of the LD to the assembly of supramolecular actin structures in real time by total internal reflection fluorescence (TIRF) microscopy. Our data shows that while LD on its own cannot polymerize under filament forming conditions, it is able to incorporate into growing F-actin filaments. This incorporation of LD triggers the formation of X-shaped filament assemblies with barbed ends that are pointing in the same direction in the majority of cases. Similarly, an increased frequency of junction sites was observed when filaments were assembled in the presence of oxidized actin. This data suggests that a disulfide bridge between Cys374 residues might stabilize LD-contacts. Based on our findings, we propose two possible models for the molecular mechanism underlying the supramolecular actin patterning in LD-related structures.
Assuntos
Citoesqueleto de Actina/química , Actinas/química , Citoesqueleto/ultraestrutura , Multimerização Proteica , Citoesqueleto de Actina/ultraestrutura , Actinas/ultraestrutura , Animais , Núcleo Celular/química , Núcleo Celular/ultraestrutura , Cisteína/química , Citoesqueleto/química , Microscopia Eletrônica de Transmissão e Varredura , Microscopia de Fluorescência , Conformação Proteica , CoelhosRESUMO
Self-assembly refers to the spontaneous organization of individual building blocks into higher order structures. It occurs in biological systems such as spherical viruses, which utilize icosahedral symmetry as a guiding principle for the assembly of coat proteins into a capsid shell. In this study, we characterize the self-assembling protein nanoparticle (SAPN) system, which was inspired by such viruses. To facilitate self-assembly, monomeric building blocks have been designed to contain two oligomerization domains. An N-terminal pentameric coiled-coil domain is linked to a C-terminal coiled-coil trimer by two glycine residues. By combining monomers with inherent propensity to form five- and threefold symmetries in higher order agglomerates, the supposition is that nanoparticles will form that exhibit local and global symmetry axes of order 3 and 5. This article explores the principles that govern the assembly of such a system. Specifically, we show that the system predominantly forms according to a spherical core-shell morphology using a combination of scanning transmission electron microscopy and small angle neutron scattering. We introduce a mathematical toolkit to provide a specific description of the possible SAPN morphologies, and we apply it to characterize all particles with maximal symmetry. In particular, we present schematics that define the relative positions of all individual chains in the symmetric SAPN particles, and provide a guide of how this approach can be generalized to nonspherical morphologies, hence providing unprecedented insights into their geometries that can be exploited in future applications.
Assuntos
Nanopartículas/química , Peptídeos/química , Multimerização Proteica , Motivos de Aminoácidos , Sequência de Aminoácidos , Modelos Teóricos , Dados de Sequência Molecular , Nanopartículas/ultraestrutura , Domínios ProteicosRESUMO
The cholesteryl ester transfer protein (CETP) enables the transfer of cholesteryl ester (CE) from high-density lipoproteins (HDL) to low-density lipoproteins (LDL) in the plasma compartment. CETP inhibition raises plasma levels of HDL cholesterol; a ternary tunnel complex with CETP bridging HDL and LDL was suggested as a mechanism. Here, we test whether the inhibition of CETP tunnel complex formation is a promising approach to suppress CE transfer from HDL to LDL, for potential treatment of cardio-vascular disease (CVD). Three monoclonal antibodies against different epitopes of CETP are assayed for their potential to interfere with CE transfer between HDL and/or LDL. Surprisingly, antibodies that target the tips of the elongated CETP molecule, interaction sites sterically required to form the suggested transfer complexes, do not interfere with CETP activity, but an antibody binding to the central region does. We show that CETP interacts with HDL, but not with LDL. Our findings demonstrate that a ternary tunnel complex is not the mechanistic prerequisite to transfer CE among lipoproteins.
Assuntos
Proteínas de Transferência de Ésteres de Colesterol/metabolismo , Ésteres do Colesterol/metabolismo , Epitopos/química , Lipoproteínas HDL/metabolismo , Lipoproteínas LDL/metabolismo , Anticorpos Monoclonais/química , Anticorpos Monoclonais/isolamento & purificação , Transporte Biológico , Linhagem Celular , Proteínas de Transferência de Ésteres de Colesterol/genética , Proteínas de Transferência de Ésteres de Colesterol/ultraestrutura , Epitopos/ultraestrutura , Expressão Gênica , Humanos , Lipoproteínas HDL/ultraestrutura , Lipoproteínas LDL/ultraestrutura , Microscopia Eletrônica de Transmissão , Ligação Proteica , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Proteínas Recombinantes/ultraestruturaRESUMO
Bilins in complex with specific proteins play key roles in many forms of life. Biliproteins have also been isolated from insects; however, structural details are rare and possible functions largely unknown. Recently, we identified a high-molecular weight biliprotein from a moth, Cerura vinula, as an arylphorin-type hexameric storage protein linked to a novel farnesyl biliverdin IXα; its unusual structure suggests formation by cleavage of mitochondrial heme A. In the present study of another moth, Spodoptera littoralis, we isolated two different biliproteins. These proteins were identified as a very high-density lipoprotein (VHDL) and as vitellin, respectively, by mass spectrometric sequencing. Both proteins are associated with three different farnesyl biliverdins IXα: the one bilin isolated from C. vinula and two new structurally closely related bilins, supposed to be intermediates of heme A degradation. The different bilin composition of the two biliproteins suggests that the presumed oxidations at the farnesyl side-chain take place mainly during egg development. The egg bilins are supposedly transferred from hemolymph VHDL to vitellin in the female. Both biliproteins show strong induced circular dichroism activity compatible with a predominance of the M-conformation of the bilins. This conformation is opposite to that of the arylphorin-type biliprotein from C. vinula. Electron microscopy of the VHDL-type biliprotein from S. littoralis provided a preliminary view of its structure as a homodimer and confirmed the biochemically determined molecular mass of â¼350 kDa. Further, images of S. littoralis hexamerins revealed a 2 × 3 construction identical to that known from the hexamerin from C. vinula.
Assuntos
Biliverdina/metabolismo , Proteínas de Transporte/metabolismo , Heme/análogos & derivados , Lipoproteínas HDL/metabolismo , Vitelinas/metabolismo , Animais , Biliverdina/química , Farneseno Álcool/química , Feminino , Heme/metabolismo , Hemolinfa/metabolismo , Larva/metabolismo , Óvulo/metabolismo , Spodoptera/metabolismoRESUMO
Successful vaccine development remains a huge challenge for infectious diseases such as malaria, HIV and influenza. As a novel way to present antigenic epitopes to the immune system, we have developed icosahedral self-assembling protein nanoparticles (SAPNs) to serve as a prototypical vaccine platform for infectious diseases. Here we examine some biophysical factors that affect the self-assembly of these nanoparticles, which have as basic building blocks coiled-coil oligomerization domains joined by a short linker region. Relying on in silico computer modeling predictions, we selected five different linker regions from the RCSB protein database that connect oligomerization domains, and then further studied the self-assembly and stability of in vitro produced nanoparticles through biophysical characterization of formed particles. One design in particular, T2i88, revealed excellent self-assembly and homogeneity thus paving the way toward a more optimized nanoparticle for vaccine applications. FROM THE CLINICAL EDITOR: Despite the widespread use of vaccines worldwide, successful development of vaccines against some diseases remains a challenge still. In this article, the authors investigated the physic-chemical and biological properties of icosahedral self-assembling protein nanoparticles (SAPNs), which mimic viral particles, in order to utilize this technology as potential platform for future design of vaccines.
Assuntos
Nanopartículas/uso terapêutico , Proteínas/imunologia , Vacinas/imunologia , Simulação por Computador , Bases de Dados de Proteínas , Infecções por HIV/imunologia , Infecções por HIV/prevenção & controle , Humanos , Influenza Humana/imunologia , Influenza Humana/prevenção & controle , Malária/imunologia , Malária/prevenção & controle , Proteínas/química , Proteínas/uso terapêutico , Vacinas/uso terapêuticoRESUMO
BACKGROUND: New experimental methods must be developed to study interaction networks in systems biology. To reduce biological noise, individual subjects, such as single cells, should be analyzed using high throughput approaches. The measurement of several correlative physical properties would further improve data consistency. Accordingly, a considerable quantity of data must be acquired, correlated, catalogued and stored in a database for subsequent analysis. RESULTS: We have developed openBEB (open Biological Experiment Browser), a software framework for data acquisition, coordination, annotation and synchronization with database solutions such as openBIS. OpenBEB consists of two main parts: A core program and a plug-in manager. Whereas the data-type independent core of openBEB maintains a local container of raw-data and metadata and provides annotation and data management tools, all data-specific tasks are performed by plug-ins. The open architecture of openBEB enables the fast integration of plug-ins, e.g., for data acquisition or visualization. A macro-interpreter allows the automation and coordination of the different modules. An update and deployment mechanism keeps the core program, the plug-ins and the metadata definition files in sync with a central repository. CONCLUSIONS: The versatility, the simple deployment and update mechanism, and the scalability in terms of module integration offered by openBEB make this software interesting for a large scientific community. OpenBEB targets three types of researcher, ideally working closely together: (i) Engineers and scientists developing new methods and instruments, e.g., for systems-biology, (ii) scientists performing biological experiments, (iii) theoreticians and mathematicians analyzing data. The design of openBEB enables the rapid development of plug-ins, which will inherently benefit from the "house keeping" abilities of the core program. We report the use of openBEB to combine live cell microscopy, microfluidic control and visual proteomics. In this example, measurements from diverse complementary techniques are combined and correlated.
Assuntos
Design de Software , Biologia de Sistemas/métodos , Automação Laboratorial , Simulação por ComputadorRESUMO
The type III secretion system machinery, also known as the injectisome, delivers bacterial effector proteins into eukaryotic cells during infection. The outer membrane YscC secretin is a major part of Yersinia enterocolitica's injectisome and is among the first components to assemble, solely assisted by its pilotin, YscW. We have determined the three-dimensional structures of the native complex and its protease-resistant core to 12 Å resolution by cryo-electron microscopy (cryo-EM) and show that YscC forms a dodecameric complex. Cryo-EM of YscC reconstituted into proteoliposomes defines the secretin's membrane-spanning region. Native YscC consists of an outer membrane ring connected via a thin cylindrical wall to a conical, periplasmic region that exposes N-terminal petals connected by flexible linkers. These petals harbor the binding site of YscD, a component of the inner membrane ring. A change in their orientation adapts the length of the YscC secretin and facilitates its interaction with YscD.
Assuntos
Proteínas de Bactérias/química , Secretina/química , Tripsina/metabolismo , Yersinia enterocolitica/metabolismo , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Bicamadas Lipídicas/química , Modelos Moleculares , Pressão Osmótica , Conformação Proteica , Multimerização Proteica , Secretina/genética , Secretina/metabolismoRESUMO
The cross-ß amyloid form of peptides and proteins represents an archetypal and widely accessible structure consisting of ordered arrays of ß-sheet filaments. These complex aggregates have remarkable chemical and physical properties, and the conversion of normally soluble functional forms of proteins into amyloid structures is linked to many debilitating human diseases, including several common forms of age-related dementia. Despite their importance, however, cross-ß amyloid fibrils have proved to be recalcitrant to detailed structural analysis. By combining structural constraints from a series of experimental techniques spanning five orders of magnitude in length scale--including magic angle spinning nuclear magnetic resonance spectroscopy, X-ray fiber diffraction, cryoelectron microscopy, scanning transmission electron microscopy, and atomic force microscopy--we report the atomic-resolution (0.5 Å) structures of three amyloid polymorphs formed by an 11-residue peptide. These structures reveal the details of the packing interactions by which the constituent ß-strands are assembled hierarchically into protofilaments, filaments, and mature fibrils.
Assuntos
Amiloide/química , Amiloide/ultraestrutura , Modelos Moleculares , Estrutura Secundária de Proteína , Microscopia Crioeletrônica , Espectroscopia de Ressonância Magnética/métodos , Microscopia Eletrônica de Transmissão e Varredura , Difração de Raios XRESUMO
A versatile methodology for electron microscopy (EM) grid preparation enabling total content sample analysis is presented. A microfluidic-dialysis conditioning module to desalt or mix samples with negative stain solution is used, combined with a robotic writing table to micro-pattern the EM grids. The method allows heterogeneous samples of minute volumes to be processed at physiological pH for structure and mass analysis, and allows the preparation characteristics to be finely tuned.
Assuntos
Microfluídica/instrumentação , Microscopia Eletrônica de Transmissão e Varredura/métodos , Animais , Células Cultivadas , Cricetinae , Coloração e Rotulagem , Biologia de Sistemas/métodosRESUMO
The Maurice E. Müller Institute, embedded in the infrastructure of the Biozentrum, University of Basel, was founded in 1985 and financed by the Maurice E. Müller Foundation of Switzerland. For 26 years its two founders, Ueli Aebi and Andreas Engel, pursued the vision of integrated structural biology. This paper reviews selected publications issuing from the Maurice E. Müller Institute for Structural Biology and marks the end of this era.
Assuntos
Academias e Institutos , Biologia Molecular/história , Biologia Molecular/métodos , Actinas/ultraestrutura , Animais , História do Século XX , Humanos , Filamentos Intermediários/ultraestrutura , Proteínas de Membrana/ultraestrutura , Microscopia de Força Atômica/métodos , Microscopia Eletrônica de Transmissão e Varredura/métodos , Membrana Nuclear/ultraestrutura , SuíçaRESUMO
Nanoparticles show great promise as potent vaccine candidates since they are readily taken up by the antigen presenting cells of the immune system. The particle size and the density of the B cell epitopes on the surface of the particles greatly influences the strength of the humoral immune response. We have developed a novel type of nanoparticle composed of peptide building blocks (Raman et al., 2006) and have used such particles to design vaccines against malaria and SARS (Kaba et al., 2009; Pimentel et al., 2009). Here we investigate the biophysical properties and the refolding conditions of a prototype of these self-assembling polypeptide nanoparticles (SAPNs). SAPNs are formed from a peptide containing a pentameric and a trimeric coiled-coil domain. At near physiological conditions the peptide self-assembles into about 27 nm, roughly spherical SAPNs. The average size of the SAPNs increases with the salt concentration. The optimal pH for their formation is between 7.5 and 8.5, while aggregation occurs at lower and higher values. A glycerol concentration of about 5% v/v is required for the formation of SAPNs with regular spherical shapes. These studies will help to optimize the immunological properties of SAPNs.
Assuntos
Antígenos/química , Nanopartículas/química , Peptídeos/química , Redobramento de Proteína , Sequência de Aminoácidos , Dicroísmo Circular/métodos , Epitopos de Linfócito B/metabolismo , Concentração de Íons de Hidrogênio , Microscopia Eletrônica de Transmissão/métodos , Modelos Moleculares , Dados de Sequência Molecular , Tamanho da Partícula , Estrutura Terciária de ProteínaRESUMO
The process of vision is initiated when the G protein-coupled receptor, rhodopsin (Rho), absorbs a photon and transitions to its activated Rho(∗) form. Rho(∗) binds the heterotrimeric G protein, transducin (G(t)) inducing GDP to GTP exchange and G(t) dissociation. Using nucleotide depletion and affinity chromatography, we trapped and purified the resulting nucleotide-free Rho(∗)·G(t) complex. Quantitative SDS-PAGE suggested a 2:1 molar ratio of Rho(∗) to G(t) in the complex and its mass determined by scanning transmission electron microscopy was 221±12kDa. A 21.6Å structure was calculated from projections of negatively stained Rho(∗)·G(t) complexes. The molecular envelope thus determined accommodated two Rho molecules together with one G(t) heterotrimer, corroborating the heteropentameric structure of the Rho(∗)·G(t) complex.
Assuntos
Complexos Multiproteicos/química , Rodopsina/química , Transducina/química , Eletroforese em Gel de Poliacrilamida , Guanosina Difosfato/química , Guanosina Trifosfato/química , Luz , Microscopia Eletrônica de Transmissão , Modelos Moleculares , Complexos Multiproteicos/ultraestrutura , Ligação Proteica , Multimerização Proteica , Rodopsina/ultraestrutura , Transducina/ultraestruturaRESUMO
Chlorosomes, the antenna complexes of green bacteria, are unique antenna systems in which pigments are organized in aggregates. Studies on isolated chlorosomes from Chlorobaculum tepidum based on SDS-PAGE, immunoblotting and molecular biology have revealed that they contain ten chlorosomal proteins, but no comprehensive information is available about the protein composition of the entire organelle. To extend these studies, chlorosomes were isolated from C. tepidum using three related and one independent isolation protocol and characterized by absorption spectroscopy, tricine SDS-PAGE, dynamic light scattering (DLS) and electron microscopy. Tricine SDS-PAGE showed the presence of more than 20 proteins with molecular weights ranging between 6 and 70 kDa. The chlorosomes varied in size. Their hydrodynamic radius (R(h) ) ranged from 51 to 75 nm and electron microscopy indicated that they were on average 140 nm wide and 170 nm long. Furthermore, the mass of 184 whole chlorosome organelles determined by scanning transmission electron microscopy ranged from 27 to 237 MDa being on average 88 (±28) MDa. In contrast their mass-per-area was independent of their size, indicating that there is a strict limit to chlorosome thickness. The average protein composition of the C. tepidum chlorosome organelles was obtained by MS/MS-driven proteomics and for the first time a detailed protein catalogue of the isolated chlorosomal proteome is presented. Based on the proteomics results for chlorosomes isolated by different protocols, four proteins that are involved in the electron or ion transport are proposed to be tightly associated with or incorporated into C. tepidum chlorosomes as well as the ten Csm proteins known to date.
Assuntos
Proteínas de Bactérias/química , Chlorobi/química , Chlorobi/citologia , Espectrometria de Massas/métodos , Organelas/química , Organelas/ultraestrutura , Proteômica/métodos , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Eletroforese em Gel de Poliacrilamida/métodos , Luz , Microscopia Eletrônica/métodos , Dados de Sequência Molecular , Proteoma/análiseRESUMO
The self-assembly of specific proteins to form insoluble amyloid fibrils is a characteristic feature of a number of age-related and debilitating diseases. Lipid-free human apolipoprotein C-II (apoC-II) forms characteristic amyloid fibrils and is one of several apolipoproteins that accumulate in amyloid deposits located within atherosclerotic plaques. X-ray diffraction analysis of aligned apoC-II fibrils indicated a simple cross-ß-structure composed of two parallel ß-sheets. Examination of apoC-II fibrils using transmission electron microscopy, scanning transmission electron microscopy, and atomic force microscopy indicated that the fibrils are flat ribbons composed of one apoC-II molecule per 4.7-Å rise of the cross-ß-structure. Cross-linking results using single-cysteine substitution mutants are consistent with a parallel in-register structural model for apoC-II fibrils. Fluorescence resonance energy transfer analysis of apoC-II fibrils labeled with specific fluorophores provided distance constraints for selected donor-acceptor pairs located within the fibrils. These findings were used to develop a simple 'letter-G-like' ß-strand-loop-ß-strand model for apoC-II fibrils. Fully solvated all-atom molecular dynamics (MD) simulations showed that the model contained a stable cross-ß-core with a flexible connecting loop devoid of persistent secondary structure. The time course of the MD simulations revealed that charge clusters in the fibril rearrange to minimize the effects of same-charge interactions inherent in parallel in-register models. Our structural model for apoC-II fibrils suggests that apoC-II monomers fold and self-assemble to form a stable cross-ß-scaffold containing relatively unstructured connecting loops.
Assuntos
Amiloide/química , Apolipoproteína C-II/química , Modelos Químicos , Simulação de Dinâmica Molecular , Sequência de Aminoácidos , Amiloide/ultraestrutura , Humanos , Microscopia de Força Atômica , Dados de Sequência Molecular , Estrutura Secundária de Proteína , Análise de Sequência de Proteína , Difração de Raios XRESUMO
Amyloid fibrils are filamentous protein aggregates implicated in several common diseases such as Alzheimer's disease and type II diabetes. Similar structures are also the molecular principle of the infectious spongiform encephalopathies such as Creutzfeldt-Jakob disease in humans, scrapie in sheep, and of the so-called yeast prions, inherited non-chromosomal elements found in yeast and fungi. Scanning transmission electron microscopy (STEM) is often used to delineate the assembly mechanism and structural properties of amyloid aggregates. In this review we consider specifically contributions and limitations of STEM for the investigation of amyloid assembly pathways, fibril polymorphisms and structural models of amyloid fibrils. This type of microscopy provides the only method to directly measure the mass-per-length (MPL) of individual filaments. Made on both in vitro assembled and ex vivo samples, STEM mass measurements have illuminated the hierarchical relationships between amyloid fibrils and revealed that polymorphic fibrils and various globular oligomers can assemble simultaneously from a single polypeptide. The MPLs also impose strong constraints on possible packing schemes, assisting in molecular model building when combined with high-resolution methods like solid-state nuclear magnetic resonance (NMR) and electron paramagnetic resonance (EPR).
Assuntos
Amiloide/química , Amiloide/ultraestrutura , Demência/patologia , Microscopia Eletrônica de Transmissão e Varredura/métodos , Modelos Moleculares , Fenótipo , HumanosRESUMO
The scanning transmission electron microscope (STEM) and the atomic force microscope (AFM) have provided a wealth of useful information on a wide variety of biological structures. These instruments have in common that they raster-scan a probe over a sample and are able to address single molecules. In the STEM the probe is a focused electron beam that is deflected by the scan-coils. Detectors collecting the scattered electrons provide quantitative information for each sub-nanometer sized sample volume irradiated. These electron scattering data can be reconstituted to images of single macromolecules or can be integrated to provide the mass of the macromolecules. Samples need to be dehydrated for such quantitative STEM imaging. In contrast, the AFM raster-scans a sharp tip over a sample surface submerged in a buffer solution to acquire information on the sample's surface topography at sub-nanometer resolution. Direct observation of function-related structural changes induced by variation of temperature, pH, ionic strength, and applied force provides insight into the structure-function relationship of macromolecules. Further, the AFM allows single molecules to be addressed and quantitatively unfolded using the tip as nano-tweezers. The performance of these two scanning probe approaches is illustrated by several examples including the chaperonin GroEL, bacterial surface layers, protein crystals, and bacterial appendices.
Assuntos
Estruturas Bacterianas/ultraestrutura , Microscopia de Força Atômica , Microscopia Eletrônica de Transmissão e Varredura , Microscopia de Força Atômica/instrumentação , Microscopia de Força Atômica/métodos , Microscopia Eletrônica de Transmissão e Varredura/instrumentação , Microscopia Eletrônica de Transmissão e Varredura/métodos , Relação Estrutura-AtividadeRESUMO
The development of biomatrices for technological and biomedical applications employs self-assembled scaffolds built from short peptidic motifs. However, biopolymers composed of protein domains would offer more varied molecular frames to introduce finer and more complex functionalities in bioreactive scaffolds using bottom-up approaches. Yet, the rules governing the three-dimensional organization of protein architectures in nature are complex and poorly understood. As a result, the synthetic fabrication of ordered protein association into polymers poses major challenges to bioengineering. We have now fabricated a self-assembling protein nanofiber with predictable morphologies and amenable to bottom-up customization, where features supporting function and assembly are spatially segregated. The design was inspired by the cross-linking of titin filaments by telethonin in the muscle sarcomere. The resulting fiber is a two-protein system that has nanopatterned peptide display capabilities as shown by the recruitment of functionalized gold nanoparticles at regular intervals of â¼ 5 nm, yielding a semiregular linear array over micrometers. This polymer promises the uncomplicated display of biologically active motifs to selectively bind and organize matter in the fine nanoscale. Further, its conceptual design has high potential for controlled plurifunctionalization.
Assuntos
Nanoestruturas/química , Biblioteca de Peptídeos , Polímeros/química , Proteínas/síntese química , Sequência de Aminoácidos , Desenho de Equipamento , Análise de Falha de Equipamento , Dados de Sequência Molecular , Tamanho da Partícula , Peptídeos/químicaRESUMO
Although the pili of Gram-positive bacteria are putative virulence factors, little is known about their structure. Here we describe the molecular architecture of pilus-1 of Streptococcus pneumoniae, which is a major cause of morbidity and mortality worldwide. One major (RrgB) and two minor components (RrgA and RrgC) assemble into the pilus. Results from TEM and scanning transmission EM show that the native pili are approximately 6 nm wide, flexible filaments that can be over 1 microm long. They are formed by a single string of RrgB monomers and have a polarity defined by nose-like protrusions. These protrusions correlate to the shape of monomeric RrgB-His, which like RrgA-His and RrgC-His has an elongated, multi-domain structure. RrgA and RrgC are only present at the opposite ends of the pilus shaft, compatible with their putative roles as adhesin and anchor to the cell wall surface, respectively. Our structural analyses provide the first direct experimental evidence that the native S. pneumoniae pilus shaft is composed exclusively of covalently linked monomeric RrgB subunits oriented head-to-tail.
Assuntos
Fímbrias Bacterianas/metabolismo , Proteínas Recombinantes/química , Streptococcus pneumoniae/metabolismo , Animais , Aderência Bacteriana , Proteínas de Bactérias/química , Western Blotting , Eletroforese em Gel de Poliacrilamida , Escherichia coli/metabolismo , Camundongos , Microscopia Eletrônica de Varredura/métodos , Microscopia Eletrônica de Transmissão/métodos , Mutação , Conformação ProteicaRESUMO
Attempts to explore the structure and function of Kch, a putative potassium channel of Escherichia coli have yielded varying results; potassium-associated functions have been found in vivo but not in vitro. Here the kch gene is shown to produce two proteins, full-length Kch and the large C-terminal cytosolic domain (the RCK domain). Further, these two proteins are associated at the initial stages of purification. Previous structural studies of full-length Kch claim that the isolated protein forms large aggregates that are not suitable for analysis. The results presented here show that the purified protein sample, although heterogeneous, has one major population with a mass of about 400kDa, implying the presence of two Kch tetramers in a complex form. A three dimensional reconstruction at 25A based on electron microscopy data from negatively stained particles, revealed a 210A long and 95A wide complex in which the two tetrameric Kch units are linked by their RCK domains, giving rise to a large central ring of density. The formation of this dimer of tetramers on expression or during purification, may explain why attempts to reconstitute Kch into liposomes for activity measurements have failed.