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1.
Adv Exp Med Biol ; 3234: 89-107, 2024.
Artigo em Inglês | MEDLINE | ID: mdl-38507202

RESUMO

Cellular machines formed by the interaction and assembly of macromolecules are essential in many processes of the living cell. These assemblies involve homo- and hetero-associations, including protein-protein, protein-DNA, protein-RNA, and protein-polysaccharide associations, most of which are reversible. This chapter describes the use of analytical ultracentrifugation, light scattering, and fluorescence-based methods, well-established biophysical techniques, to characterize interactions leading to the formation of macromolecular complexes and their modulation in response to specific or unspecific factors. We also illustrate, with several examples taken from studies on bacterial processes, the advantages of the combined use of subsets of these techniques as orthogonal analytical methods to analyze protein oligomerization and polymerization, interactions with ligands, hetero-associations involving membrane proteins, and protein-nucleic acid complexes.


Assuntos
Proteínas , RNA , Espectrometria de Fluorescência , Proteínas/química , Substâncias Macromoleculares , Ultracentrifugação/métodos
2.
Adv Exp Med Biol ; 3234: 109-123, 2024.
Artigo em Inglês | MEDLINE | ID: mdl-38507203

RESUMO

Nuclear magnetic resonance (NMR) and native mass spectrometry (MS) are mature physicochemical techniques with long histories and important applications. NMR spectroscopy provides detailed information about the structure, dynamics, interactions, and chemical environment of biomolecules. MS is an effective approach for determining the mass of biomolecules with high accuracy, sensitivity, and speed. The two techniques offer unique advantages and provide solid tools for structural biology. In the present review, we discuss their individual merits in the context of their applications to structural studies in biology with specific focus on protein interactions and evaluate their limitations. We provide specific examples in which these techniques can complement each other, providing new information on the same scientific case. We discuss how the field may develop and what challenges are expected in the future. Overall, the combination of NMR and MS plays an increasingly important role in integrative structural biology, assisting scientists in deciphering the three-dimensional structure of composite macromolecular assemblies.


Assuntos
Imageamento por Ressonância Magnética , Espectrometria de Massas/métodos , Espectroscopia de Ressonância Magnética , Substâncias Macromoleculares/química , Ressonância Magnética Nuclear Biomolecular/métodos
3.
Adv Exp Med Biol ; 3234: 125-140, 2024.
Artigo em Inglês | MEDLINE | ID: mdl-38507204

RESUMO

X-ray crystallography has for most of the last century been the standard technique to determine the high-resolution structure of biological macromolecules, including multi-subunit protein-protein and protein-nucleic acids as large as the ribosome and viruses. As such, the successful application of X-ray crystallography to many biological problems revolutionized biology and biomedicine by solving the structures of small molecules and vitamins, peptides and proteins, DNA and RNA molecules, and many complexes-affording a detailed knowledge of the structures that clarified biological and chemical mechanisms, conformational changes, interactions, catalysis and the biological processes underlying DNA replication, translation, and protein synthesis. Now reaching well into the first quarter of the twenty-first century, X-ray crystallography shares the structural biology stage with cryo-electron microscopy and other innovative structure determination methods, as relevant and central to our understanding of biological function and structure as ever. In this chapter, we provide an overview of modern X-ray crystallography and how it interfaces with other mainstream structural biology techniques, with an emphasis on macromolecular complexes.


Assuntos
Biologia Molecular , Proteínas , Cristalografia por Raios X , Microscopia Crioeletrônica/métodos , Proteínas/química , Substâncias Macromoleculares/química
4.
Adv Exp Med Biol ; 3234: 163-172, 2024.
Artigo em Inglês | MEDLINE | ID: mdl-38507206

RESUMO

Small angle X-ray scattering (SAXS) is a versatile technique that can provide unique insights in the solution structure of macromolecules and their complexes, covering the size range from small peptides to complete viral assemblies. Technological and conceptual advances in the last two decades have tremendously improved the accessibility of the technique and transformed it into an indispensable tool for structural biology. In this chapter we introduce and discuss several approaches to collecting SAXS data on macromolecular complexes, including several approaches to online chromatography. We include practical advice on experimental design and point out common pitfalls of the technique.


Assuntos
Cromatografia , Espalhamento a Baixo Ângulo , Raios X , Difração de Raios X , Substâncias Macromoleculares/química
5.
Adv Exp Med Biol ; 3234: 173-190, 2024.
Artigo em Inglês | MEDLINE | ID: mdl-38507207

RESUMO

High-resolution structure determination by electron cryo-microscopy underwent a step change in recent years. This now allows study of challenging samples which previously were inaccessible for structure determination, including membrane proteins. These developments shift the focus in the field to the next bottlenecks which are high-quality sample preparations. While the amounts of sample required for cryo-EM are relatively small, sample quality is the key challenge. Sample quality is influenced by the stability of complexes which depends on buffer composition, inherent flexibility of the sample, and the method of solubilization from the membrane for membrane proteins. It further depends on the choice of sample support, grid pre-treatment and cryo-grid freezing protocol. Here, we discuss various widely applicable approaches to improve sample quality for structural analysis by cryo-EM.


Assuntos
Elétrons , Proteínas de Membrana , Microscopia Crioeletrônica/métodos , Congelamento , Manejo de Espécimes/métodos , Substâncias Macromoleculares
6.
Adv Exp Med Biol ; 3234: 191-205, 2024.
Artigo em Inglês | MEDLINE | ID: mdl-38507208

RESUMO

Recent advancements in cryo-electron microscopy (cryo-TEM) have enabled the determination of structures of macromolecular complexes at near-atomic resolution, establishing it as a pivotal tool in Structural Biology. This high resolution allows for the detection of ligands and substrates under physiological conditions. Enhancements in detectors and imaging devices, like phase plates, improve signal quality, facilitating the reconstruction of even smaller macromolecular complexes. The 100-kDa barrier has been surpassed, presenting new opportunities for pharmacological research and expanding the scope of crystallographic analyses in the pharmaceutical industry. Cryo-TEM produces vast data sets from minimal samples, and refined classification methods can identify different conformational states of macromolecular complexes, offering deeper insights into the functional characteristics of macromolecular systems. Additionally, cryo-TEM is paving the way for time-resolved microscopy, with rapid freezing techniques capturing snapshots of vital structural changes in biological complexes. Finally, in Structural Cell Biology, advanced cryo-TEM, through tomographic procedures, is revealing conformational changes related to the specific subcellular localization of macromolecular systems and their interactions within cells.


Assuntos
Biologia Molecular , Microscopia Crioeletrônica/métodos , Conformação Molecular , Substâncias Macromoleculares/química
7.
Nat Commun ; 15(1): 2265, 2024 Mar 13.
Artigo em Inglês | MEDLINE | ID: mdl-38480681

RESUMO

Rational design of next-generation therapeutics can be facilitated by high-resolution structures of drug targets bound to small-molecule inhibitors. However, application of structure-based methods to macromolecules refractory to crystallization has been hampered by the often-limiting resolution and throughput of cryogenic electron microscopy (cryo-EM). Here, we use high-resolution cryo-EM to determine structures of the CDK-activating kinase, a master regulator of cell growth and division, in its free and nucleotide-bound states and in complex with 15 inhibitors at up to 1.8 Å resolution. Our structures provide detailed insight into inhibitor interactions and networks of water molecules in the active site of cyclin-dependent kinase 7 and provide insights into the mechanisms contributing to inhibitor selectivity, thereby providing the basis for rational design of next-generation therapeutics. These results establish a methodological framework for the use of high-resolution cryo-EM in structure-based drug design.


Assuntos
Quinase Ativadora de Quinase Dependente de Ciclina , Desenho de Fármacos , Humanos , Microscopia Crioeletrônica/métodos , Substâncias Macromoleculares/química , Ciclo Celular
8.
Molecules ; 29(5)2024 Feb 24.
Artigo em Inglês | MEDLINE | ID: mdl-38474502

RESUMO

Enzymes play an important role in numerous natural processes and are increasingly being utilized as environmentally friendly substitutes and alternatives to many common catalysts. Their essential advantages are high catalytic efficiency, substrate specificity, minimal formation of byproducts, and low energy demand. All of these benefits make enzymes highly desirable targets of academic research and industrial development. This review has the modest aim of briefly overviewing the classification, mechanism of action, basic kinetics and reaction condition effects that are common across all six enzyme classes. Special attention is devoted to immobilization strategies as the main tools to improve the resistance to environmental stress factors (temperature, pH and solvents) and prolong the catalytic lifecycle of these biocatalysts. The advantages and drawbacks of methods such as macromolecular crosslinking, solid scaffold carriers, entrapment, and surface modification (covalent and physical) are discussed and illustrated using numerous examples. Among the hundreds and possibly thousands of known and recently discovered enzymes, hydrolases and oxidoreductases are distinguished by their relative availability, stability, and wide use in synthetic applications, which include pharmaceutics, food and beverage treatments, environmental clean-up, and polymerizations. Two representatives of those groups-laccase (an oxidoreductase) and lipase (a hydrolase)-are discussed at length, including their structure, catalytic mechanism, and diverse usage. Objective representation of the current status and emerging trends are provided in the main conclusions.


Assuntos
Lacase , Lipase , Lipase/química , Lacase/química , Enzimas Imobilizadas/química , Catálise , Substâncias Macromoleculares
9.
Int J Biol Macromol ; 263(Pt 2): 129939, 2024 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-38423909

RESUMO

Polysaccharides are biologically essential macromolecules, widely exist in plants, which are used in food, medicine, bioactives' encapsulation, targeted delivery and other fields. Suitable extraction technology can not only improve the yield, but also regulate the physicochemical, improve the functional property, and is the basis for the research and application of polysaccharide. High pressure (HP) extraction (HPE) induces the breakage of raw material cells and tissues through rapid changes in pressure, increases extraction yield, reduces extraction time, and modifies structure of polysaccharides. However, thus far, literature review on the mechanism of extraction, improved yield and modified structure of HPE polysaccharide is lacking. Therefore, the present work reviews the mechanism of HPE polysaccharide, increasing extraction yield, regulating physicochemical and functional properties, modifying structure and improving activity. This review contributes to a full understanding of the HPE or development of polysaccharide production and modification methods and promotes the application of HP technology in polysaccharide production.


Assuntos
Medicina , Polissacarídeos , Polissacarídeos/farmacologia , Polissacarídeos/química , Extratos Vegetais/química , Substâncias Macromoleculares , Antioxidantes/química
10.
Protein Sci ; 33(3): e4909, 2024 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-38358136

RESUMO

A flat mask-based model is almost universally used in macromolecular crystallography to account for disordered (bulk) solvent. This model assumes any voxel of the crystal unit cell that is not occupied by the atomic model is occupied by the solvent. The properties of this solvent are assumed to be exactly the same across the whole volume of the unit cell. While this is a reasonable approximation in practice, there are a number of scenarios where this model becomes suboptimal. In this work, we enumerate several of these scenarios and describe a new generalized approach to modeling the bulk-solvent which we refer to as mosaic bulk-solvent model. The mosaic bulk-solvent model allows nonuniform features of the solvent in the crystal to be accounted for in a computationally efficient way. It is implemented in the computational crystallography toolbox and the Phenix software.


Assuntos
Software , Solventes/química , Cristalografia por Raios X , Substâncias Macromoleculares/química
11.
Acta Crystallogr D Struct Biol ; 80(Pt 3): 148-158, 2024 Mar 01.
Artigo em Inglês | MEDLINE | ID: mdl-38411552

RESUMO

The validation of structural models obtained by macromolecular X-ray crystallography against experimental diffraction data, whether before deposition into the PDB or after, is typically carried out exclusively against the merged data that are eventually archived along with the atomic coordinates. It is shown here that the availability of unmerged reflection data enables valuable additional analyses to be performed that yield improvements in the final models, and tools are presented to implement them, together with examples of the results to which they give access. The first example is the automatic identification and removal of image ranges affected by loss of crystal centering or by excessive decay of the diffraction pattern as a result of radiation damage. The second example is the `reflection-auditing' process, whereby individual merged data items showing especially poor agreement with model predictions during refinement are investigated thanks to the specific metadata (such as image number and detector position) that are available for the corresponding unmerged data, potentially revealing previously undiagnosed instrumental, experimental or processing problems. The third example is the calculation of so-called F(early) - F(late) maps from carefully selected subsets of unmerged amplitude data, which can not only highlight the location and extent of radiation damage but can also provide guidance towards suitable fine-grained parametrizations to model the localized effects of such damage.


Assuntos
Cristalografia por Raios X , Substâncias Macromoleculares/química
12.
Langmuir ; 40(9): 4615-4622, 2024 03 05.
Artigo em Inglês | MEDLINE | ID: mdl-38387073

RESUMO

The cellular environment is crowded with macromolecules of different shapes and sizes. The effect of this macromolecular crowding has been studied in a variety of synthetic crowding environments: two popular examples are the compact colloid-like Ficoll macromolecule and the globular protein bovine serum albumin (BSA). Recent studies have indicated that a significant component of bound or surface-associated water in these crowders reduces the available free volume. In this work, Brillouin light scattering experiments were performed on aqueous solutions of Ficoll 70 and Ficoll 400 with concentrations ranging from 1 to 35 wt % and BSA with concentrations of 1 to 27 wt %. From the dependence of spectral peak parameters on polymer concentration, we determined fundamental solution properties: hypersound velocity, adiabatic bulk modulus and compressibility, apparent viscosity, and hypersound attenuation. The existing theory that ignores intermolecular interactions can capture only the observed linear trends in the frequency shift up to a threshold concentration, beyond which a quadratic term accounting for intermolecular interactions is necessary. This likely indicates a transition from the dilute to semidilute regime. In the Ficoll solutions (but not BSA), we see evidence for a central mode, which is indicative of relaxation in the hydration shell of Ficoll.


Assuntos
Soroalbumina Bovina , Água , Ficoll/química , Soroalbumina Bovina/química , Substâncias Macromoleculares , Análise Espectral , Soluções/química
13.
Bioinformatics ; 40(3)2024 Mar 04.
Artigo em Inglês | MEDLINE | ID: mdl-38391029

RESUMO

MOTIVATION: Integrative structural modeling combines data from experiments, physical principles, statistics of previous structures, and prior models to obtain structures of macromolecular assemblies that are challenging to characterize experimentally. The choice of model representation is a key decision in integrative modeling, as it dictates the accuracy of scoring, efficiency of sampling, and resolution of analysis. But currently, the choice is usually made ad hoc, manually. RESULTS: Here, we report NestOR (Nested Sampling for Optimizing Representation), a fully automated, statistically rigorous method based on Bayesian model selection to identify the optimal coarse-grained representation for a given integrative modeling setup. Given an integrative modeling setup, it determines the optimal representations from given candidate representations based on their model evidence and sampling efficiency. The performance of NestOR was evaluated on a benchmark of four macromolecular assemblies. AVAILABILITY AND IMPLEMENTATION: NestOR is implemented in the Integrative Modeling Platform (https://integrativemodeling.org) and is available at https://github.com/isblab/nestor. Data for the benchmark is at https://www.doi.org/10.5281/zenodo.10360718.


Assuntos
Benchmarking , Teorema de Bayes , Substâncias Macromoleculares/química
14.
Cell ; 187(3): 513-516, 2024 Feb 01.
Artigo em Inglês | MEDLINE | ID: mdl-38306977

RESUMO

In November 2023, structural biologists from different countries and different disciplines gathered at the Cell Symposium: Structural biology from the nanoscale to cellular mesoscale to discuss recent breakthroughs, including structures of proteins and macromolecular complexes in a cellular context as well as virus structures obtained by using different techniques. At the symposium, Cell editor Jia Cheng and Karin Kühnel, editor-in-chief of Structure, spoke with Drs. Beili Wu, Mingjie Zhang, and Zihe Rao about their experiences doing structural biology research in China and about their perspectives for the future. An edited transcript of the conversation is presented below, and the full conversation is available with the article online.


Assuntos
Biologia Molecular , Substâncias Macromoleculares , China
15.
Cell ; 187(3): 545-562, 2024 Feb 01.
Artigo em Inglês | MEDLINE | ID: mdl-38306981

RESUMO

Determining the structure and mechanisms of all individual functional modules of cells at high molecular detail has often been seen as equal to understanding how cells work. Recent technical advances have led to a flush of high-resolution structures of various macromolecular machines, but despite this wealth of detailed information, our understanding of cellular function remains incomplete. Here, we discuss present-day limitations of structural biology and highlight novel technologies that may enable us to analyze molecular functions directly inside cells. We predict that the progression toward structural cell biology will involve a shift toward conceptualizing a 4D virtual reality of cells using digital twins. These will capture cellular segments in a highly enriched molecular detail, include dynamic changes, and facilitate simulations of molecular processes, leading to novel and experimentally testable predictions. Transferring biological questions into algorithms that learn from the existing wealth of data and explore novel solutions may ultimately unveil how cells work.


Assuntos
Biologia , Biologia Computacional , Substâncias Macromoleculares/química
16.
Protein Sci ; 33(3): e4894, 2024 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-38358134

RESUMO

α-synuclein is an intrinsically disordered protein (IDP) whose aggregation in presynaptic neuronal cells is a pathological hallmark of Lewy body formation and Parkinson's disease. This aggregation process is likely affected by the crowded macromolecular cellular environment. In this study, α-synuclein was studied in the presence of both a synthetic crowder, Ficoll70, and a biological crowder composed of lysed cells that better mimics the biocomplexity of the cellular environment. 15 N-1 H HSQC NMR results show similar α-synuclein chemical shifts in non-crowded and all crowded conditions implying that it remains similarly unstructured in all conditions. Nevertheless, both HSQC NMR and fluorescence measurements indicate that, only in the cell lysate, α-synuclein forms aggregates over a timescale of 48 h. 15 N-edited diffusion measurements indicate that all crowders slow down the α-synuclein's diffusivity. Interestingly, at high concentrations, α-synuclein diffuses faster in cell lysate than in Ficoll70, possibly due to additional soft (e.g., electrostatic or hydrophobic) interactions. 15 N-edited relaxation measurements show that some residues are more mobile in cell lysate than in Ficoll70; the rates that are most different are predominantly in hydrophobic residues. We thus examined cell lysates with reduced hydrophobicity and found slower dynamics (higher relaxation rates) in several α-synuclein residues. Taken together, these experiments suggest that while cell lysate does not substantially affect α-synuclein structure (HSQC spectra), it does affect chain dynamics and translational diffusion, and strongly affects aggregation over a timescale of days, in a manner that is different from either no crowder or an artificial crowder: soft hydrophobic interactions are implicated.


Assuntos
Proteínas Intrinsicamente Desordenadas , Doença de Parkinson , Humanos , alfa-Sinucleína/química , Doença de Parkinson/metabolismo , Conformação Proteica , Substâncias Macromoleculares/química , Proteínas Intrinsicamente Desordenadas/química
17.
J Phys Chem B ; 128(6): 1394-1406, 2024 Feb 15.
Artigo em Inglês | MEDLINE | ID: mdl-38316012

RESUMO

Molecular simulations of biomacromolecules that assemble into multimeric complexes remain a challenge due to computationally inaccessible length and time scales. Low-resolution and implicit-solvent coarse-grained modeling approaches using traditional nonbonded interactions (both pairwise and spherically isotropic) have been able to partially address this gap. However, these models may fail to capture the complex anisotropic interactions present at macromolecular interfaces unless higher-order interaction potentials are incorporated at the expense of the computational cost. In this work, we introduce an alternate and systematic approach to represent directional interactions at protein-protein interfaces by using virtual sites restricted to pairwise interactions. We show that virtual site interaction parameters can be optimized within a relative entropy minimization framework by using only information from known statistics between coarse-grained sites. We compare our virtual site models to traditional coarse-grained models using two case studies of multimeric protein assemblies and find that the virtual site models predict pairwise correlations with higher fidelity and, more importantly, assembly behavior that is morphologically consistent with experiments. Our study underscores the importance of anisotropic interaction representations and paves the way for more accurate yet computationally efficient coarse-grained simulations of macromolecular assembly in future research.


Assuntos
Proteínas , Solventes , Entropia , Substâncias Macromoleculares
18.
BMC Bioinformatics ; 25(1): 77, 2024 Feb 20.
Artigo em Inglês | MEDLINE | ID: mdl-38378489

RESUMO

BACKGROUND: Cryo-electron microscopy (Cryo-EM) plays an increasingly important role in the determination of the three-dimensional (3D) structure of macromolecules. In order to achieve 3D reconstruction results close to atomic resolution, 2D single-particle image classification is not only conducive to single-particle selection, but also a key step that affects 3D reconstruction. The main task is to cluster and align 2D single-grain images into non-heterogeneous groups to obtain sharper single-grain images by averaging calculations. The main difficulties are that the cryo-EM single-particle image has a low signal-to-noise ratio (SNR), cannot manually label the data, and the projection direction is random and the distribution is unknown. Therefore, in the low SNR scenario, how to obtain the characteristic information of the effective particles, improve the clustering accuracy, and thus improve the reconstruction accuracy, is a key problem in the 2D image analysis of single particles of cryo-EM. RESULTS: Aiming at the above problems, we propose a learnable deep clustering method and a fast alignment weighted averaging method based on frequency domain space to effectively improve the class averaging results and improve the reconstruction accuracy. In particular, it is very prominent in the feature extraction and dimensionality reduction module. Compared with the classification method based on Bayesian and great likelihood, a large amount of single particle data is required to estimate the relative angle orientation of macromolecular single particles in the 3D structure, and we propose that the clustering method shows good results. CONCLUSIONS: SimcryoCluster can use the contrastive learning method to perform well in the unlabeled high-noise cryo-EM single particle image classification task, making it an important tool for cryo-EM protein structure determination.


Assuntos
Processamento de Imagem Assistida por Computador , Semântica , Microscopia Crioeletrônica/métodos , Teorema de Bayes , Processamento de Imagem Assistida por Computador/métodos , Análise por Conglomerados , Substâncias Macromoleculares
19.
Nano Lett ; 24(8): 2457-2464, 2024 Feb 28.
Artigo em Inglês | MEDLINE | ID: mdl-38373157

RESUMO

The ability of living objects to respond rapidly en masse to various stimuli or stress is an important function in response to externally applied changes in the local environment. This occurs across many length scales, for instance, bacteria swarming in response to different stimuli or stress and macromolecular crowding within cells. Currently there are few mechanisms to induce similar autonomous behaviors within populations of synthetic protocells. Herein, we report a system in which populations of individual objects behave in a coordinated manner in response to changes in the energetic environment by the emergent self-organization of large object swarms. These swarms contain protocell populations of approximately 60 000 individuals. We demonstrate the dissipative nature of the hierarchical constructs, which persist under appropriate UV stimulation. Finally, we identify the ability of the object populations to change behaviors in an adaptive population-wide response to the local energetic environment.


Assuntos
Células Artificiais , Humanos , Substâncias Macromoleculares
20.
J Phys Chem B ; 128(8): 1876-1883, 2024 Feb 29.
Artigo em Inglês | MEDLINE | ID: mdl-38355410

RESUMO

Genetic sequencing is a vital process that requires the transport of charged nucleic acids through transmembrane nanopores. Single-molecule studies show that macromolecular bulk crowding facilitates the capture of these polymers, leading to a high throughput of nanopore sensors. Motivated by these observations, a minimal discrete-state stochastic framework was developed to describe the role of poly(ethylene glycol) (PEG) crowders in varying concentrations in the transport of ssDNA through α-hemolysin nanopores. This theory suggested that the cooperative partitioning of polycationic PEGs controls the capture of ssDNA due to underlying electrostatic interactions. Herein, we investigate the impact of the size variation of PEGs on the capture event. Even though larger crowders attract ssDNA strongly to enhance its capture, our results show that considerable cooperative partitioning of PEGs is also required to achieve high interevent frequency. The exact analytical results are supported by existing single-molecule studies. Since real cellular conditions are heterogeneous, its influence on the ssDNA capture rate is studied by introducing a binary mixture of crowders. Our results indicate that the "polymer-pushing-polymer" concept possibly affects the capture rate depending on the mixture composition. These new findings provide valuable insights into the microscopic mechanism of the capture process, which eventually allows for accurate genome sequencing in crowded solutions.


Assuntos
Nanoporos , Nanoporos/ultraestrutura , DNA de Cadeia Simples , Polímeros , Substâncias Macromoleculares , Polietilenoglicóis
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