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1.
Nat Commun ; 12(1): 1957, 2021 03 30.
Artigo em Inglês | MEDLINE | ID: mdl-33785757

RESUMO

Tomographic reconstruction of cryopreserved specimens imaged in an electron microscope followed by extraction and averaging of sub-volumes has been successfully used to derive atomic models of macromolecules in their biological environment. Eliminating biochemical isolation steps required by other techniques, this method opens up the cell to in-situ structural studies. However, the need to compensate for errors in targeting introduced during mechanical navigation of the specimen significantly slows down tomographic data collection thus limiting its practical value. Here, we introduce protocols for tilt-series acquisition and processing that accelerate data collection speed by up to an order of magnitude and improve map resolution compared to existing approaches. We achieve this by using beam-image shift to multiply the number of areas imaged at each stage position, by integrating geometrical constraints during imaging to achieve high precision targeting, and by performing per-tilt astigmatic CTF estimation and data-driven exposure weighting to improve final map resolution. We validated our beam image-shift electron cryo-tomography (BISECT) approach by determining the structure of a low molecular weight target (~300 kDa) at 3.6 Å resolution where density for individual side chains is clearly resolved.


Assuntos
Microscopia Crioeletrônica/métodos , Tomografia com Microscopia Eletrônica/métodos , Processamento de Imagem Assistida por Computador/métodos , Tomografia Computadorizada por Raios X/métodos , Algoritmos , Imageamento Tridimensional/métodos , Substâncias Macromoleculares/química , Substâncias Macromoleculares/ultraestrutura , Tamanho da Partícula , Reprodutibilidade dos Testes
2.
Food Chem ; 352: 129343, 2021 Aug 01.
Artigo em Inglês | MEDLINE | ID: mdl-33652194

RESUMO

In this work, the interaction of wine macromolecules with a bovine serum albumin (BSA) was investigated using fluorescence correlation spectroscopy (FCS). FCS offers the opportunity to study molecular and macromolecular aggregation without disturbing the wine by introducing only very small amounts of fluorescently labelled molecules to the system. It was observed that the diffusion coefficient of fluorescently labelled BSA varies with the addition of wine macromolecules, indicating changes in the protein conformation and the formation of complexes and aggregates. The addition of a wine polysaccharide rhamnogalacturonan II-enriched fraction led to aggregation, while addition of a mannoprotein-enriched fraction exhibited a protective effect on protein aggregation. Proteins strongly interacted with tannins, leading to the precipitation of protein-tannin complexes, while the presence of polysaccharides prevented this precipitation. Finally, the application of FCS was demonstrated in real wines, to investigate the problem of protein haze formation through live monitoring of heat-induced aggregation in wine.


Assuntos
Análise de Alimentos , Substâncias Macromoleculares/química , Espectrometria de Fluorescência , Vinho/análise
3.
Molecules ; 26(5)2021 Feb 25.
Artigo em Inglês | MEDLINE | ID: mdl-33668767

RESUMO

The self-recognition and self-assembly of biomolecules are spontaneous processes that occur in Nature and allow the formation of ordered structures, at the nanoscale or even at the macroscale, under thermodynamic and kinetic equilibrium as a consequence of specific and local interactions. In particular, peptides and peptidomimetics play an elected role, as they may allow a rational approach to elucidate biological mechanisms to develop new drugs, biomaterials, catalysts, or semiconductors. The forces that rule self-recognition and self-assembly processes are weak interactions, such as hydrogen bonding, electrostatic attractions, and van der Waals forces, and they underlie the formation of the secondary structure (e.g., α-helix, ß-sheet, polyproline II helix), which plays a key role in all biological processes. Here, we present recent and significant examples whereby design was successfully applied to attain the desired structural motifs toward function. These studies are important to understand the main interactions ruling the biological processes and the onset of many pathologies. The types of secondary structure adopted by peptides during self-assembly have a fundamental importance not only on the type of nano- or macro-structure formed but also on the properties of biomaterials, such as the types of interaction, encapsulation, non-covalent interaction, or covalent interaction, which are ultimately useful for applications in drug delivery.


Assuntos
Materiais Biocompatíveis/química , Desenho de Fármacos , Peptídeos/química , Proteínas/química , Sistemas de Liberação de Medicamentos , Substâncias Macromoleculares/química
4.
Molecules ; 26(4)2021 Feb 11.
Artigo em Inglês | MEDLINE | ID: mdl-33670156

RESUMO

Recently, metal-coordinated orthogonal self-assembly has been used as a feasible and efficient method in the construction of polymeric materials, which can also provide supramolecular self-assembly complexes with different topologies. Herein, a cryptand with a rigid pyridyl group on the third arm derived from BMP32C10 was synthesized. Through coordination-driven self-assembly with a bidentate organoplatinum(II) acceptor or tetradentate Pd(BF4)2•4CH3CN, a di-cryptand complex and tetra-cryptand complex were prepared, respectively. Subsequently, through the addition of a di-paraquat guest, linear and cross-linked supramolecular polymers were constructed through orthogonal self-assembly, respectively. By comparing their proton nuclear magnetic resonance (1H NMR) and diffusion-ordered spectroscopy (DOSY) spectra, it was found that the degrees of polymerization were dependent not only on the concentrations of the monomers but also on the topologies of the supramolecular polymers.


Assuntos
Complexos de Coordenação/química , Éteres Cíclicos/química , Metais/química , Paraquat/química , Bases de Schiff/química , Substâncias Macromoleculares/química , Estrutura Molecular , Polímeros/química
5.
J Vis Exp ; (169)2021 03 13.
Artigo em Inglês | MEDLINE | ID: mdl-33779594

RESUMO

The use of neutron macromolecular crystallography (NMX) is expanding rapidly with most structures determined in the last decade thanks to new NMX beamlines having been built and increased availability of structure refinement software. However, the neutron sources currently available for NMX are significantly weaker than equivalent sources for X-ray crystallography. Despite advances in this field, significantly larger crystals will always be required for neutron diffraction studies, particularly with the tendency to study ever-larger macromolecules and complexes. Further improvements in methods and instrumentation suited to growing larger crystals are therefore necessary for the use of NMX to expand. In this work, we introduce rational strategies and a crystal growth bench (OptiCrys) developed in our laboratory that combines real-time observation through a microscope-mounted video camera with precise automated control of crystallization solutions (e.g., precipitant concentration, pH, additive, temperature). We then demonstrate how this control of temperature and chemical composition facilitates the search for optimal crystallization conditions using model soluble proteins. Thorough knowledge of the crystallization phase diagram is crucial for selecting the starting position and the kinetic path for any crystallization experiment. We show how a rational approach can control the size and number of crystals generated based on knowledge of multidimensional phase diagrams.


Assuntos
Cristalização/métodos , Substâncias Macromoleculares/química , Difração de Nêutrons/métodos , Nêutrons , Proteínas/química , Cristalografia por Raios X , Humanos
6.
Molecules ; 26(4)2021 Feb 07.
Artigo em Inglês | MEDLINE | ID: mdl-33562215

RESUMO

Therapeutic proteins, such as growth factors (GFs), have been used in tissue engineering (TE) approaches for their ability to provide signals to cells and orchestrate the formation of functional tissue. However, to be effective and minimize off-target effects, GFs should be delivered at the target site with temporal control. In addition, protein drugs are typically sensitive water soluble macromolecules with delicate structure. As such, hydrogels, containing large amounts of water, provide a compatible environment for the direct incorporation of proteins within the hydrogel network, while their release rate can be tuned by engineering the network chemistry and density. Being formed by transient crosslinks, afforded by non-covalent interactions, supramolecular hydrogels offer important advantages for protein delivery applications. This review describes various types of supramolecular hydrogels using a repertoire of diverse building blocks, their use for protein delivery and their further application in TE contexts. By reviewing the recent literature on this topic, the merits of supramolecular hydrogels are highlighted as well as their limitations, with high expectations for new advances they will provide for TE in the near future.


Assuntos
Hidrogéis/química , Peptídeos e Proteínas de Sinalização Intercelular/química , Proteínas/química , Engenharia Tecidual , Humanos , Hidrogéis/uso terapêutico , Peptídeos e Proteínas de Sinalização Intercelular/uso terapêutico , Substâncias Macromoleculares/química , Substâncias Macromoleculares/uso terapêutico , Proteínas/uso terapêutico , Água/química
7.
Int J Nanomedicine ; 16: 1261-1280, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-33628020

RESUMO

Diabetes mellitus is a major threat to human health. Both its incidence and prevalence have been rising steadily over the past few decades. Biomacromolecular agents such as insulin and glucagon-like peptide 1 receptor agonists are commonly used hypoglycemic drugs that play important roles in the treatment of diabetes. However, their traditional frequent administration may cause numerous side effects, such as pain, infection or local tissue necrosis. To address these issues, many novel subcutaneous delivery systems have been developed in recent years. In this review, we survey recent developments in subcutaneous delivery systems of biomacromolecular hypoglycemic drugs, including sustained-release delivery systems and stimuli-responsive delivery systems, and summarize the advantages and limitations of these systems. Future opportunities and challenges are discussed as well.


Assuntos
Diabetes Mellitus/tratamento farmacológico , Sistemas de Liberação de Medicamentos , Substâncias Macromoleculares/química , Tela Subcutânea/fisiologia , Animais , Humanos , Hipoglicemiantes/química , Hipoglicemiantes/uso terapêutico , Nanopartículas/química
8.
Nat Methods ; 18(2): 176-185, 2021 02.
Artigo em Inglês | MEDLINE | ID: mdl-33542510

RESUMO

Cryo-electron microscopy (cryo-EM) single-particle analysis has proven powerful in determining the structures of rigid macromolecules. However, many imaged protein complexes exhibit conformational and compositional heterogeneity that poses a major challenge to existing three-dimensional reconstruction methods. Here, we present cryoDRGN, an algorithm that leverages the representation power of deep neural networks to directly reconstruct continuous distributions of 3D density maps and map per-particle heterogeneity of single-particle cryo-EM datasets. Using cryoDRGN, we uncovered residual heterogeneity in high-resolution datasets of the 80S ribosome and the RAG complex, revealed a new structural state of the assembling 50S ribosome, and visualized large-scale continuous motions of a spliceosome complex. CryoDRGN contains interactive tools to visualize a dataset's distribution of per-particle variability, generate density maps for exploratory analysis, extract particle subsets for use with other tools and generate trajectories to visualize molecular motions. CryoDRGN is open-source software freely available at http://cryodrgn.csail.mit.edu .


Assuntos
Microscopia Crioeletrônica/métodos , Substâncias Macromoleculares/química , Redes Neurais de Computação , Estrutura Molecular
9.
Eur J Med Chem ; 212: 113105, 2021 Feb 15.
Artigo em Inglês | MEDLINE | ID: mdl-33385835

RESUMO

Macro biomolecules are of vital importance in regulating the biofunctions in organisms, in which proteins (including peptides when mentioned below) and nucleic acids (NAs) are the most important. Therefore, these proteins and NAs can be applied as "drugs" to regulate the biofunctions from abnormal to normal. Either for proteins and NAs, the most challenging thing is to avoid the biodegradation or physicochemical degradation before they reach the targeted location, and then functions as complete functional structures. Hence, appropriate delivery systems are very important which can protect them from these degradations. Cyclodextrins (CDs) based delivery systems achieved mega successes due to their outstanding pharmaceutical properties and there have been several reviews on CDs based small molecule drug delivery systems recently. But for biomolecules, which are getting more and more important for modern therapies, however, there are very few reviews to systematically summarize and analyze the CDs-based macro biomolecules delivery systems, especially for proteins. In this review, there were some of the notable examples were summarized for the macro biomolecules (proteins and NAs) delivery based on CDs. For proteins, this review included insulin, lysozyme, bovine serum albumin (BSA), green fluorescent protein (GFP) and IgG's, etc. deliveries in slow release, stimulating responsive release or targeting release manners. For NAs, this review summarized cationic CD-polymers and CD-cluster monomers as NAs carriers, notably, including the multicomponents targeting CD-based carriers and the virus-like RNA assembly method siRNA carriers.


Assuntos
Ciclodextrinas/química , Sistemas de Liberação de Medicamentos , Ácidos Nucleicos/química , Peptídeos/química , Proteínas/química , Portadores de Fármacos/química , Humanos , Substâncias Macromoleculares/química
10.
Int J Biol Macromol ; 170: 728-750, 2021 Feb 15.
Artigo em Inglês | MEDLINE | ID: mdl-33387543

RESUMO

Hydrogels are widely used for wound healing applications due to their similarity to the native extracellular matrix (ECM) and ability to provide a moist environment. However, lack of multifunctionality and low mechanical properties of previously developed hydrogels may limit their ability to support skin tissue regeneration. Incorporating various biomaterials and nanostructures into the hydrogels is an emerging approach to develop multifunctional hydrogels with new functions that are beneficial for wound healing. These multifunctional hydrogels can be fabricated with a wide range of functions and properties, including antibacterial, antioxidant, bioadhesive, and appropriate mechanical properties. Two approaches can be used for development of multifunctional hydrogel-based dressings; taking the advantages of the chemical composition of biomaterials and addition of nanomaterials or nanostructures. A large number of synthetic and natural polymers, bioactive molecules, or nanomaterials have been used to obtain hydrogel-based dressings with multifunctionality for wound healing applications. In the present review paper, advances in the development of multifunctional hydrogel-based dressings for wound healing have been highlighted.


Assuntos
Materiais Biocompatíveis/química , Materiais Biocompatíveis/farmacologia , Hidrogéis/química , Hidrogéis/farmacologia , Substâncias Macromoleculares/química , Substâncias Macromoleculares/farmacologia , Cicatrização/efeitos dos fármacos , Animais , Humanos , Polímeros/química
11.
Nat Chem ; 13(2): 131-139, 2021 02.
Artigo em Inglês | MEDLINE | ID: mdl-33514936

RESUMO

Pharmaceutical drug therapy is often hindered by issues caused by poor drug selectivity, including unwanted side effects and drug resistance. Spatial and temporal control over drug activation in response to stimuli is a promising strategy to attenuate and circumvent these problems. Here we use ultrasound to activate drugs from inactive macromolecules or nano-assemblies through the controlled scission of mechanochemically labile covalent bonds and weak non-covalent bonds. We show that a polymer with a disulfide motif at the centre of the main chain releases an alkaloid-based anticancer drug from its ß-carbonate linker by a force-induced intramolecular 5-exo-trig cyclization. Second, aminoglycoside antibiotics complexed by a multi-aptamer RNA structure are activated by the mechanochemical opening and scission of the nucleic acid backbone. Lastly, nanoparticle-polymer and nanoparticle-nanoparticle assemblies held together by hydrogen bonds between the peptide antibiotic vancomycin and its complementary peptide target are activated by force-induced scission of hydrogen bonds. This work demonstrates the potential of ultrasound to activate mechanoresponsive prodrug systems.


Assuntos
Ativação Metabólica/fisiologia , Sistemas de Liberação de Medicamentos/métodos , Pró-Fármacos/química , Dissulfetos/química , Células HeLa , Humanos , Ligação de Hidrogênio , Substâncias Macromoleculares/química , Estrutura Molecular , Peptídeos/química , Polímeros/química , Ondas Ultrassônicas
12.
Nat Rev Mol Cell Biol ; 22(3): 196-213, 2021 03.
Artigo em Inglês | MEDLINE | ID: mdl-33510441

RESUMO

Biomolecular condensates are membraneless intracellular assemblies that often form via liquid-liquid phase separation and have the ability to concentrate biopolymers. Research over the past 10 years has revealed that condensates play fundamental roles in cellular organization and physiology, and our understanding of the molecular principles, components and forces underlying their formation has substantially increased. Condensate assembly is tightly regulated in the intracellular environment, and failure to control condensate properties, formation and dissolution can lead to protein misfolding and aggregation, which are often the cause of ageing-associated diseases. In this Review, we describe the mechanisms and regulation of condensate assembly and dissolution, highlight recent advances in understanding the role of biomolecular condensates in ageing and disease, and discuss how cellular stress, ageing-related loss of homeostasis and a decline in protein quality control may contribute to the formation of aberrant, disease-causing condensates. Our improved understanding of condensate pathology provides a promising path for the treatment of protein aggregation diseases.


Assuntos
Envelhecimento , Substâncias Macromoleculares/química , Complexos Multiproteicos/fisiologia , Agregação Patológica de Proteínas/etiologia , Estresse Fisiológico/fisiologia , Envelhecimento/metabolismo , Envelhecimento/patologia , Animais , Fenômenos Fisiológicos Celulares , Humanos , Substâncias Macromoleculares/metabolismo , Agregados Proteicos/fisiologia , Agregação Patológica de Proteínas/metabolismo
13.
J Phys Chem Lett ; 12(1): 633-641, 2021 Jan 14.
Artigo em Inglês | MEDLINE | ID: mdl-33382941

RESUMO

Computer simulation approaches in biomolecular recognition processes have come a long way. In this Perspective, we highlight a series of recent success stories in which computer simulations have played a remarkable role in elucidating the atomic resolution mechanism of kinetic processes of protein-ligand binding in a quantitative fashion. In particular, we show that a robust combination of unbiased simulation, harnessed by a high-fidelity computing environment, and Markov state modeling approaches has been instrumental in revealing novel protein-ligand recognition pathways in multiple systems. We also elucidate the role of recent developments in enhanced sampling approaches in providing the much-needed impetus in accelerating simulation of the ligand recognition process. We identify multiple key issues, including force fields and the sampling bottleneck, which are currently preventing the field from achieving quantitative reconstruction of experimental measurements. Finally, we suggest a possible way forward via adoption of multiscale approaches and coarse-grained simulations as next steps toward efficient elucidation of ligand binding kinetics.


Assuntos
Simulação por Computador , Substâncias Macromoleculares/metabolismo , Substâncias Macromoleculares/química , Simulação de Dinâmica Molecular
14.
Methods Mol Biol ; 2215: 83-111, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-33368000

RESUMO

Electron cryo-tomography (cryo-ET) is a technique that allows the investigation of intact macromolecular complexes while they are in their cellular milieu. Over the years, cryo-ET has had a huge impact on our understanding of how large biomolecular complexes look like, how they assemble, disassemble, function, and evolve(d). Recent hardware and software developments and combining cryo-ET with other techniques, e.g., focused ion beam milling (FIB-milling) and cryo-light microscopy, has extended the realm of cryo-ET to include transient molecular complexes embedded deep in thick samples (like eukaryotic cells) and enhanced the resolution of structures obtained by cryo-ET. In this chapter, we will present an outline of how to perform cryo-ET studies on a wide variety of biological samples including prokaryotic and eukaryotic cells and biological plant tissues. This outline will include sample preparation, data collection, and data processing as well as hybrid approaches like FIB-milling, cryosectioning, and cryo-correlated light and electron microscopy (cryo-CLEM).


Assuntos
Microscopia Crioeletrônica/métodos , Tomografia com Microscopia Eletrônica/métodos , Substâncias Macromoleculares/química , Células 3T3 , Animais , Arabidopsis/citologia , Arabidopsis/metabolismo , Arabidopsis/ultraestrutura , Crioultramicrotomia , Tomografia com Microscopia Eletrônica/instrumentação , Fibroblastos/citologia , Fibroblastos/metabolismo , Fibroblastos/ultraestrutura , Humanos , Camundongos , Software , Manejo de Espécimes
15.
Methods Mol Biol ; 2215: 125-144, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-33368002

RESUMO

Illuminating a specimen with a parallel electron beam is critical for many experiments in transmission electron microscopy as deviations from this condition cause considerable deterioration of image quality. Carefully establishing parallel illumination is particularly important on two-condenser lens transmission electron microscopes (TEMs) as the parallel illumination condition is limited to a single beam intensity value on these instruments. It was recently shown that a Thermo Fisher Scientific Talos Arctica, a two-condenser lens TEM operating at 200 kV, equipped with a Gatan K2 Summit direct electron detector is capable of resolving frozen-hydrated macromolecules of various sizes and internal symmetries to better than 3 Å resolution using single particle methodologies. A critical aspect of the success of these findings was the careful alignment of the electron microscope to ensure the specimen was illuminated with a parallel electron beam. Here, this chapter describes how to establish parallel illumination conditions in a Talos Arctica TEM for high-resolution cryogenic data collection for structure determination.


Assuntos
Substâncias Macromoleculares/química , Microscopia Eletrônica de Transmissão/instrumentação , Processamento de Imagem Assistida por Computador , Imagem Individual de Molécula , Software
16.
Methods Mol Biol ; 2215: 145-160, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-33368003

RESUMO

Single-particle analysis of electron cryo-microscopy (cryo-EM) images allows structure determination of macromolecular complexes. But when these molecules adopt many different conformations, traditional image processing approaches often lead to blurred reconstructions. By considering complexes to be comprised of multiple, independently moving rigid bodies, multi-body refinement in RELION enables structure determination of highly flexible complexes, while at the same time providing a characterization of the motions in the complex. Here, we describe how to perform multi-body refinement in RELION using a publicly available example. We outline how to prepare the necessary files, how to run the actual multi-body calculation, and how to interpret its output. This method can be applied to any cryo-EM data set of flexible complexes that can be divided into two or more bodies, each with a minimum molecular weight of 100-150 kDa.


Assuntos
Processamento de Imagem Assistida por Computador/métodos , Substâncias Macromoleculares/química , Imagem Individual de Molécula/métodos , Microscopia Crioeletrônica , Modelos Moleculares , Conformação Molecular , Software
17.
Methods Mol Biol ; 2192: 243-268, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-33230778

RESUMO

Cryo-electron tomography (cryo-ET) enables the three-dimensional (3D) visualization of macromolecular complexes in their native environment (in situ). The ability to visualize macromolecules in situ is in particular advantageous for complex, membrane-associated processes, such as mitochondrial translation. Mitochondrial translation occurs almost exclusively associated with the inner mitochondrial membrane, giving rise to the mitochondrial DNA-encoded subunits of oxidative phosphorylation machinery. In cryo-ET, the 3D volume is reconstructed from a set of 2D projections of a frozen-hydrated specimen, which is sequentially tilted and imaged at different angles in a transmission electron microscope. In combination with subtomogram analysis, cryo-ET enables the structure determination of macromolecular complexes and their 3D organization. In this chapter, we summarize all steps required for structural characterization of mitochondrial ribosomes in situ, ranging from data acquisition to tomogram reconstruction and subtomogram analysis.


Assuntos
Microscopia Crioeletrônica/métodos , Tomografia com Microscopia Eletrônica/métodos , Mitocôndrias/metabolismo , Biossíntese de Proteínas/fisiologia , Células HEK293 , Humanos , Processamento de Imagem Assistida por Computador/métodos , Imageamento Tridimensional/métodos , Substâncias Macromoleculares/química , Software
18.
Methods Mol Biol ; 2247: 125-143, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-33301115

RESUMO

Interactions between protein complexes and DNA are central regulators of the cell life. They control the activation and inactivation of a large set of nuclear processes including transcription, replication, recombination, repair, and chromosome structures. In the literature, protein-DNA interactions are characterized by highly complementary approaches including large-scale studies and analyses in cells. Biophysical approaches with purified materials help to evaluate if these interactions are direct or not. They provide quantitative information on the strength and specificity of the interactions between proteins or protein complexes and their DNA substrates. Isothermal titration calorimetry (ITC) and microscale thermophoresis (MST) are widely used and are complementary methods to characterize nucleo-protein complexes and quantitatively measure protein-DNA interactions. We present here protocols to analyze the interactions between a DNA repair complex, Ku70-Ku80 (Ku) (154 kDa), and DNA substrates. ITC is a label-free method performed with both partners in solution. It serves to determine the dissociation constant (Kd), the enthalpy (ΔH), and the stoichiometry N of an interaction. MST is used to measure the Kd between the protein or the DNA labeled with a fluorescent probe. We report the data obtained on Ku-DNA interactions with ITC and MST and discuss advantages and drawbacks of both the methods.


Assuntos
Proteínas de Ligação a DNA/química , DNA/química , Substâncias Macromoleculares/química , Fenômenos Bioquímicos , Calorimetria , DNA/genética , DNA/metabolismo , Proteínas de Ligação a DNA/metabolismo , Modelos Moleculares , Conformação Molecular , Ligação Proteica , Relação Estrutura-Atividade , Termodinâmica
19.
Methods Mol Biol ; 2247: 221-241, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-33301120

RESUMO

Mass spectrometry (MS)-based strategies have emerged as key elements for structural modeling of proteins and their assemblies. In particular, merging together complementary MS tools, through the so-called hybrid approaches, has enabled structural characterization of proteins in their near-native states. Here, we describe how different MS techniques, such as native MS, chemical cross-linking MS, and ion mobility MS, are brought together using sophisticated computational algorithms and modeling restraints. We demonstrate the applicability of the strategy by building accurate models of multimeric protein assemblies. These strategies can practically be applied to any protein complex of interest and be readily integrated with other structural approaches such as electron density maps from cryo-electron microscopy.


Assuntos
Substâncias Macromoleculares/química , Espectrometria de Massas , Modelos Moleculares , Sequência de Aminoácidos , Microscopia Crioeletrônica/métodos , Espectrometria de Massas/métodos , Conformação Molecular , Estrutura Molecular , Conformação Proteica , Proteínas/química
20.
Methods Mol Biol ; 2247: 243-256, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-33301121

RESUMO

Electron microscopy is a powerful tool for studying the homogeneity and structure of biomolecular complexes. The small wavelength of electron and the availability of electron optics enable the direct visualization of macromolecular assemblies in a large range of sizes between 5 and 100 nm. This informs us about the degree of multimerization or aggregation and provides precise information about their general shape and dimensions. When combined with sophisticated image analysis protocols, three-dimensional (3D) information can be gained from 2D projections of the sample, leading to a structural description. When intermediate steps of a reaction can be imaged, insights into the mode of action of macromolecules can be gained, and structure-function relations can be established. However, the way the sample is prepared for its observation within the vacuum of an electron microscope determines the information that can be retrieved from the experiment. We will review two commonly used specimen preparation protocols for subsequent single-particle electron microscopy observation, namely negative staining and vitrification.


Assuntos
Microscopia Crioeletrônica , Substâncias Macromoleculares/química , Microscopia Crioeletrônica/métodos
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