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1.
Nature ; 580(7801): 37-38, 2020 04.
Artigo em Inglês | MEDLINE | ID: mdl-32238936
2.
Science ; 368(6486): 30-31, 2020 04 03.
Artigo em Inglês | MEDLINE | ID: mdl-32241937
3.
Ultrasonics ; 103: 106086, 2020 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-32070827

RESUMO

Ultrasonic bioreactors have been used for in vitro experimentation to study cellular responses to low-intensity pulsed ultrasound. The presence of an air interface in these bioreactors contributes to variability in the acoustic pressure field, reducing experimental reproducibility. A multiphysics finite element model was developed to simulate the acoustic field in an in-dish ultrasonic bioreactor, where the transducer is immersed in culture medium above the dish surface, and the effects of replacing air below the dish in the bioreactor with a water layer bounded by an acoustic absorbent layer were evaluated. Frequency domain simulations showed that the spatially-averaged pressure at the dish surface alternated between a minimum and maximum level as the distance between the dish and transducer increased. The ratio of the maximum to minimum level was 6.5-fold when the air interface was present, and this ratio dropped to 1.8-fold with replacement of the air interface. However, radial pressure variability was present with or without the air interface in the bioreactor model. Time-dependent simulations showed that the increase in acoustic pressure to a maximum level after US signal activation and the pressure drop after signal cessation were faster when the water-coupled non-reflective layer was used to replace the air layer below the dish, generating a pressure pattern that more closely followed the applied pulsed ultrasound signal due to reduced wave reflection and interference. Overall, this work showed that having water rather than air in contact with the lower dish surface when paired with an acoustic absorbent layer resulted in a less variable pressure field, providing an improved bioreactor design for in vitro experiments.


Assuntos
Acústica , Reatores Biológicos , Análise de Elementos Finitos , Fenômenos Biofísicos , Meios de Cultura , Desenho de Equipamento , Pressão , Software , Propriedades de Superfície , Transdutores , Ultrassom , Água
4.
Nat Commun ; 11(1): 975, 2020 02 20.
Artigo em Inglês | MEDLINE | ID: mdl-32080202

RESUMO

The reliable detection of environmental molecules in the presence of noise is an important cellular function, yet the underlying computational mechanisms are not well understood. We introduce a model of two interacting sensors which allows for the principled exploration of signal statistics, cooperation strategies and the role of energy consumption in optimal sensing, quantified through the mutual information between the signal and the sensors. Here we report that in general the optimal sensing strategy depends both on the noise level and the statistics of the signals. For joint, correlated signals, energy consuming (nonequilibrium), asymmetric couplings result in maximum information gain in the low-noise, high-signal-correlation limit. Surprisingly we also find that energy consumption is not always required for optimal sensing. We generalise our model to incorporate time integration of the sensor state by a population of readout molecules, and demonstrate that sensor interaction and energy consumption remain important for optimal sensing.


Assuntos
Técnicas Biossensoriais/métodos , Fenômenos Biofísicos , Técnicas Biossensoriais/estatística & dados numéricos , Bioestatística , Células/metabolismo , Células Quimiorreceptoras/metabolismo , Metabolismo Energético , Regulação da Expressão Gênica , Modelos Biológicos , Transdução de Sinais , Razão Sinal-Ruído
5.
Phys Rev Lett ; 124(3): 038003, 2020 Jan 24.
Artigo em Inglês | MEDLINE | ID: mdl-32031851

RESUMO

Lotus leaves floating on water usually experience short-wavelength edge wrinkling that decays toward the center, while the leaves growing above water normally morph into a global bending cone shape with long rippled waves near the edge. Observations suggest that the underlying water (liquid substrate) significantly affects the morphogenesis of leaves. To understand the biophysical mechanism under such phenomena, we develop mathematical models that can effectively account for inhomogeneous differential growth of floating and freestanding leaves to quantitatively predict formation and evolution of their morphology. We find, both theoretically and experimentally, that the short-wavelength buckled configuration is energetically favorable for growing membranes lying on liquid, while the global buckling shape is more preferable for suspended ones. Other influencing factors such as the stem or vein, heterogeneity, and dimension are also investigated. Our results provide a fundamental insight into a variety of plant morphogenesis affected by water foundation and suggest that such surface instabilities can be harnessed for morphology control of biomimetic deployable structures using substrate or edge actuation.


Assuntos
Lotus/crescimento & desenvolvimento , Modelos Biológicos , Água/química , Fenômenos Biofísicos , Lotus/anatomia & histologia , Morfogênese , Folhas de Planta/anatomia & histologia , Folhas de Planta/crescimento & desenvolvimento
6.
Nucleic Acids Res ; 48(4): 1748-1763, 2020 02 28.
Artigo em Inglês | MEDLINE | ID: mdl-31930331

RESUMO

The double-helical structure of DNA results from canonical base pairing and stacking interactions. However, variations from steady-state conformations resulting from mechanical perturbations in cells have physiological relevance but their dependence on sequence remains unclear. Here, we use molecular dynamics simulations showing sequence differences result in markedly different structural motifs upon physiological twisting and stretching. We simulate overextension on different sequences of DNA ((AA)12, (AT)12, (CC)12 and (CG)12) with supercoiling densities at 200 and 50 mM salt concentrations. We find that DNA denatures in the majority of stretching simulations, surprisingly including those with over-twisted DNA. GC-rich sequences are observed to be more stable than AT-rich ones, with the specific response dependent on the base pair order. Furthermore, we find that (AT)12 forms stable periodic structures with non-canonical hydrogen bonds in some regions and non-canonical stacking in others, whereas (CG)12 forms a stacking motif of four base pairs independent of supercoiling density. Our results demonstrate that 20-30% DNA extension is sufficient for breaking B-DNA around and significantly above cellular supercoiling, and that the DNA sequence is crucial for understanding structural changes under mechanical stress. Our findings have important implications for the activities of protein machinery interacting with DNA in all cells.


Assuntos
Pareamento de Bases/genética , Sequência de Bases/genética , DNA/química , Fenômenos Biofísicos , DNA/genética , Sequência Rica em GC/genética , Ligação de Hidrogênio , Simulação de Dinâmica Molecular , Estrutura Molecular , Conformação de Ácido Nucleico
7.
Food Chem ; 312: 126064, 2020 May 15.
Artigo em Inglês | MEDLINE | ID: mdl-31891887

RESUMO

Biophysical insight into the binding interaction between the major whey protein, ß-Lactoglobulin (ßLG) and vitamin B12, was studied using different spectroscopic tools such as steady-state & time-resolved fluorescence spectroscopy, Circular Dichroism (CD) and Fluorescence Correlation Spectroscopy (FCS). The intrinsic fluorescence of ßLG was quenched by vitamin B12. From the time-resolved fluorescence experiment, the nature of quenching was found to be static suggesting ground-state complex formation between ßLG and vitamin B12, which was also supported by the excitation spectra. Synchronous fluorescence spectra revealed that the tryptophan residue microenvironment of ßLG was affected by the vitamin B12. The CD spectra suggested that the secondary structure of the ßLG remains unaffected by vitamin B12. From the FCS experiment, the tertiary structure of ßLG was observed to be stable in the presence of vitamin B12 at the single-molecule level. The outcome of this study might have potential applications in the food and pharmaceutical industry.


Assuntos
Lactoglobulinas/química , Vitamina B 12/química , Fenômenos Biofísicos , Dicroísmo Circular , Lactoglobulinas/metabolismo , Estrutura Secundária de Proteína , Espectrometria de Fluorescência , Triptofano/química , Vitamina B 12/metabolismo
8.
Nat Methods ; 17(2): 175-183, 2020 02.
Artigo em Inglês | MEDLINE | ID: mdl-31907444

RESUMO

In mammalian cells, much of signal transduction is mediated by weak protein-protein interactions between globular peptide-binding domains (PBDs) and unstructured peptidic motifs in partner proteins. The number and diversity of these PBDs (over 1,800 are known), their low binding affinities and the sensitivity of binding properties to minor sequence variation represent a substantial challenge to experimental and computational analysis of PBD specificity and the networks PBDs create. Here, we introduce a bespoke machine-learning approach, hierarchical statistical mechanical modeling (HSM), capable of accurately predicting the affinities of PBD-peptide interactions across multiple protein families. By synthesizing biophysical priors within a modern machine-learning framework, HSM outperforms existing computational methods and high-throughput experimental assays. HSM models are interpretable in familiar biophysical terms at three spatial scales: the energetics of protein-peptide binding, the multidentate organization of protein-protein interactions and the global architecture of signaling networks.


Assuntos
Aprendizado de Máquina , Peptídeos/metabolismo , Proteínas/metabolismo , Transdução de Sinais , Fenômenos Biofísicos , Humanos , Ligação Proteica , Reprodutibilidade dos Testes , Domínios de Homologia de src
9.
Proc Natl Acad Sci U S A ; 117(2): 1049-1058, 2020 01 14.
Artigo em Inglês | MEDLINE | ID: mdl-31896582

RESUMO

Targeting Clostridium difficile infection is challenging because treatment options are limited, and high recurrence rates are common. One reason for this is that hypervirulent C. difficile strains often have a binary toxin termed the C. difficile toxin, in addition to the enterotoxins TsdA and TsdB. The C. difficile toxin has an enzymatic component, termed CDTa, and a pore-forming or delivery subunit termed CDTb. CDTb was characterized here using a combination of single-particle cryoelectron microscopy, X-ray crystallography, NMR, and other biophysical methods. In the absence of CDTa, 2 di-heptamer structures for activated CDTb (1.0 MDa) were solved at atomic resolution, including a symmetric (SymCDTb; 3.14 Å) and an asymmetric form (AsymCDTb; 2.84 Å). Roles played by 2 receptor-binding domains of activated CDTb were of particular interest since the receptor-binding domain 1 lacks sequence homology to any other known toxin, and the receptor-binding domain 2 is completely absent in other well-studied heptameric toxins (i.e., anthrax). For AsymCDTb, a Ca2+ binding site was discovered in the first receptor-binding domain that is important for its stability, and the second receptor-binding domain was found to be critical for host cell toxicity and the di-heptamer fold for both forms of activated CDTb. Together, these studies represent a starting point for developing structure-based drug-design strategies to target the most severe strains of C. difficile.


Assuntos
ADP Ribose Transferases/química , ADP Ribose Transferases/metabolismo , Proteínas de Bactérias/química , Proteínas de Bactérias/metabolismo , Toxinas Bacterianas/química , Toxinas Bacterianas/metabolismo , Clostridium difficile/metabolismo , Enterotoxinas/química , Enterotoxinas/metabolismo , ADP Ribose Transferases/genética , Animais , Proteínas de Bactérias/genética , Sítios de Ligação , Fenômenos Biofísicos , Chlorocebus aethiops , Microscopia Crioeletrônica , Cristalografia por Raios X , Modelos Moleculares , Ressonância Magnética Nuclear Biomolecular , Conformação Proteica , Domínios Proteicos , Células Vero
10.
Nat Commun ; 11(1): 124, 2020 01 08.
Artigo em Inglês | MEDLINE | ID: mdl-31913281

RESUMO

Recent high-throughput transcription factor (TF) binding assays revealed that TF cooperativity is a widespread phenomenon. However, a global mechanistic and functional understanding of TF cooperativity is still lacking. To address this, here we introduce a statistical learning framework that provides structural insight into TF cooperativity and its functional consequences based on next generation sequencing data. We identify DNA shape as driver for cooperativity, with a particularly strong effect for Forkhead-Ets pairs. Follow-up experiments reveal a local shape preference at the Ets-DNA-Forkhead interface and decreased cooperativity upon loss of the interaction. Additionally, we discover many functional associations for cooperatively bound TFs. Examination of the link between FOXO1:ETV6 and lymphomas reveals that their joint expression levels improve patient clinical outcome stratification. Altogether, our results demonstrate that inter-family cooperative TF binding is driven by position-specific DNA readout mechanisms, which provides an additional regulatory layer for downstream biological functions.


Assuntos
Fatores de Transcrição/química , Fatores de Transcrição/metabolismo , Fenômenos Biofísicos , DNA/química , DNA/genética , DNA/metabolismo , Regulação da Expressão Gênica , Humanos , Cinética , Modelos Genéticos , Fenótipo , Ligação Proteica , Fatores de Transcrição/genética
11.
Biochem Soc Trans ; 48(1): 221-229, 2020 02 28.
Artigo em Inglês | MEDLINE | ID: mdl-31922188

RESUMO

Motile cilia are a striking example of the functional cellular organelle, conserved across all the eukaryotic species. Motile cilia allow the swimming of cells and small organisms and transport of liquids across epithelial tissues. Whilst the molecular structure is now very well understood, the dynamics of cilia is not well established either at the single cilium level nor at the level of collective beating. Indeed, a full understanding of this requires connecting together behaviour across various lengthscales, from the molecular to the organelle, then at the cellular level and up to the tissue scale. Aside from the fundamental interest in this system, understanding beating is important to elucidate aspects of embryonic development and a variety of health conditions from fertility to genetic and infectious diseases of the airways.


Assuntos
Fenômenos Biofísicos , Cílios/fisiologia , Flagelos/fisiologia , Organelas/fisiologia , Animais , Epitélio/fisiologia , Células Eucarióticas/fisiologia , Humanos , Hidrodinâmica , Mucosa Respiratória/fisiologia
12.
PLoS One ; 15(1): e0226544, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-31923278

RESUMO

Ocean acidification is an emerging consequence of anthropogenic carbon dioxide emissions. The full extent of the biological impacts are currently not entirely defined. However, it is expected that invertebrate species that rely on the mineral calcium carbonate will be directly affected. Despite the limited understanding of the full extent of potential impacts and responses there is a need to identify potential pathways for human societies to be affected by ocean acidification. Research on these social implications is a small but developing field. This research contributes to this field by using an impact assessment framework, informed by a biophysical model of future species distributions, to investigate potential impacts facing Atlantic Canadian society from potential changes in shellfish fisheries driven by ocean acidification and climate change. New Brunswick and Nova Scotia are expected to see declines in resource accessibility but are relatively socially insulated from these changes. Conversely, Prince Edward Island, along with Newfoundland and Labrador are more socially vulnerable to potential losses in fisheries, but are expected to experience relatively minor net changes in access.


Assuntos
Oceano Atlântico , Mudança Climática , Pesqueiros , Água do Mar/química , Fatores Socioeconômicos , Fenômenos Biofísicos , Canadá , Concentração de Íons de Hidrogênio , Modelos Teóricos
13.
Nat Commun ; 11(1): 566, 2020 Jan 28.
Artigo em Inglês | MEDLINE | ID: mdl-31992698

RESUMO

Characterizing folding and complex formation of biomolecules provides a view into their thermodynamics, kinetics and folding pathways. Deciphering kinetic intermediates is particularly important because they can often be targeted by drugs. The key advantage of native mass spectrometry over conventional methods that monitor a single observable is its ability to identify and quantify coexisting species. Here, we show the design of a temperature-jump electrospray source for mass spectrometry that allows one to perform fast kinetics experiments (0.16-32 s) at different temperatures (10-90 °C). The setup allows recording of both folding and unfolding kinetics by using temperature jumps from high to low, and low to high, temperatures. Six biological systems, ranging from peptides to proteins to DNA complexes, exemplify the use of this device. Using temperature-dependent experiments, the folding and unfolding of a DNA triplex are studied, providing detailed information on its thermodynamics and kinetics.


Assuntos
Espectrometria de Massas/métodos , Desnaturação de Ácido Nucleico , Ligação Proteica , Desnaturação Proteica , Dobramento de Proteína , Temperatura , Fenômenos Biofísicos , DNA/química , Cinética , Desdobramento de Proteína , Termodinâmica
14.
Nat Commun ; 11(1): 540, 2020 Jan 28.
Artigo em Inglês | MEDLINE | ID: mdl-31992709

RESUMO

Eukaryotic transcription factors (TF) function by binding to short 6-10 bp DNA recognition sites located near their target genes, which are scattered through vast genomes. Such process surmounts enormous specificity, efficiency and celerity challenges using a molecular mechanism that remains poorly understood. Combining biophysical experiments, theory and bioinformatics, we dissect the interplay between the DNA-binding domain of Engrailed, a Drosophila TF, and the regulatory regions of its target genes. We find that Engrailed binding affinity is strongly amplified by the DNA regions flanking the recognition site, which contain long tracts of degenerate recognition-site repeats. Such DNA organization operates as an antenna that attracts TF molecules in a promiscuous exchange among myriads of intermediate affinity binding sites. The antenna ensures a local TF supply, enables gene tracking and fine control of the target site's basal occupancy. This mechanism illuminates puzzling gene expression data and suggests novel engineering strategies to control gene expression.


Assuntos
Sítios de Ligação , DNA/metabolismo , Proteínas de Drosophila/metabolismo , Eucariotos/genética , Regulação da Expressão Gênica , Proteínas de Homeodomínio/metabolismo , Fatores de Transcrição/metabolismo , Animais , Sequência de Bases , Fenômenos Biofísicos , Biologia Computacional , Proteínas de Ligação a DNA , Drosophila melanogaster/genética , Drosophila melanogaster/metabolismo , Genoma , Cinética , Modelos Teóricos , Conformação Molecular , Termodinâmica
15.
J Forensic Sci ; 65(1): 255-258, 2020 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-31411743

RESUMO

The horizontal displacement of the human body resulting from fatal fall from a height is an important variable commonly used to inversely determine the cause or identify other forensic aspects of the fall. When examining the horizontal displacement, the wind effect is generally ignored. This technical note reports analytical modeling of the falling process, utilizing previous measurements of wind force acting on the human body, for determining the functional relationship between the wind speed and the horizontal displacement in the falling process. The result reveals that it does not take extremely rare wind conditions to cause a considerable shift of the human body, highlighting the importance to consider wind as a factor in investigations of fatal falls.


Assuntos
Acidentes por Quedas , Ciências Forenses/métodos , Modelos Biológicos , Modelos Estatísticos , Vento , Fenômenos Biofísicos , Estatura , Índice de Massa Corporal , Feminino , Humanos , Masculino
16.
Nat Commun ; 10(1): 5672, 2019 12 12.
Artigo em Inglês | MEDLINE | ID: mdl-31831746

RESUMO

Constructing colloidal particles into functional nanostructures, materials, and devices is a promising yet challenging direction. Many optical techniques have been developed to trap, manipulate, assemble, and print colloidal particles from aqueous solutions into desired configurations on solid substrates. However, these techniques operated in liquid environments generally suffer from pattern collapses, Brownian motion, and challenges that come with reconfigurable assembly. Here, we develop an all-optical technique, termed optothermally-gated photon nudging (OPN), for the versatile manipulation and dynamic patterning of a variety of colloidal particles on a solid substrate at nanoscale accuracy. OPN takes advantage of a thin surfactant layer to optothermally modulate the particle-substrate interaction, which enables the manipulation of colloidal particles on solid substrates with optical scattering force. Along with in situ optical spectroscopy, our non-invasive and contactless nanomanipulation technique will find various applications in nanofabrication, nanophotonics, nanoelectronics, and colloidal sciences.


Assuntos
Coloides/química , Nanoestruturas/química , Óptica e Fotônica/métodos , Fótons , Fenômenos Biofísicos , Movimento (Física) , Tamanho da Partícula , Tensoativos/química , Temperatura
17.
PLoS Comput Biol ; 15(12): e1006941, 2019 12.
Artigo em Inglês | MEDLINE | ID: mdl-31869343

RESUMO

Ca2+/calmodulin-dependent protein kinase II (CaMKII) accounts for up to 2 percent of all brain protein and is essential to memory function. CaMKII activity is known to regulate dynamic shifts in the size and signaling strength of neuronal connections, a process known as synaptic plasticity. Increasingly, computational models are used to explore synaptic plasticity and the mechanisms regulating CaMKII activity. Conventional modeling approaches may exclude biophysical detail due to the impractical number of state combinations that arise when explicitly monitoring the conformational changes, ligand binding, and phosphorylation events that occur on each of the CaMKII holoenzyme's subunits. To manage the combinatorial explosion without necessitating bias or loss in biological accuracy, we use a specialized syntax in the software MCell to create a rule-based model of a twelve-subunit CaMKII holoenzyme. Here we validate the rule-based model against previous experimental measures of CaMKII activity and investigate molecular mechanisms of CaMKII regulation. Specifically, we explore how Ca2+/CaM-binding may both stabilize CaMKII subunit activation and regulate maintenance of CaMKII autophosphorylation. Noting that Ca2+/CaM and protein phosphatases bind CaMKII at nearby or overlapping sites, we compare model scenarios in which Ca2+/CaM and protein phosphatase do or do not structurally exclude each other's binding to CaMKII. Our results suggest a functional mechanism for the so-called "CaM trapping" phenomenon, wherein Ca2+/CaM may structurally exclude phosphatase binding and thereby prolong CaMKII autophosphorylation. We conclude that structural protection of autophosphorylated CaMKII by Ca2+/CaM may be an important mechanism for regulation of synaptic plasticity.


Assuntos
Proteína Quinase Tipo 2 Dependente de Cálcio-Calmodulina/química , Proteína Quinase Tipo 2 Dependente de Cálcio-Calmodulina/metabolismo , Calmodulina/metabolismo , Animais , Sítios de Ligação , Fenômenos Biofísicos , Cálcio/metabolismo , Biologia Computacional , Estabilidade Enzimática , Hipocampo/metabolismo , Humanos , Modelos Moleculares , Modelos Neurológicos , Plasticidade Neuronal , Fosfoproteínas Fosfatases/metabolismo , Fosforilação , Ligação Proteica , Estrutura Quaternária de Proteína , Subunidades Proteicas
18.
Adv Exp Med Biol ; 1174: 1-33, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31713195

RESUMO

The aggregation of proteins into fibrillar structures is a central process implicated in the onset and development of several devastating neuro-degenerative diseases, but can, in contrast to these pathological roles, also fulfil important biological functions. In both scenarios, an understanding of the mechanisms by which soluble proteins convert to their fibrillar forms represents a fundamental objective for molecular sciences. This chapter details the different classes of microscopic processes responsible for this conversion and discusses how they can be described by a mathematical formulation of the aggregation kinetics. We present easily accessible experimental quantities that allow the determination of the dominant pathways of aggregation, as well as a general strategy to obtain detailed solutions to the kinetic rate laws that yield the microscopic rate constants of the individual processes of nucleation and growth. This chapter discusses a framework for a structured approach to address key questions regarding the dynamics of protein aggregation and shows how the use of chemical kinetics to tackle complex biophysical systems can lead to a deeper understanding of the underlying physical and chemical principles.


Assuntos
Fenômenos Biofísicos , Peptídeos , Cinética , Peptídeos/química , Peptídeos/metabolismo , Agregação Patológica de Proteínas , Proteínas/química , Proteínas/metabolismo
19.
Adv Exp Med Biol ; 1174: 61-112, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31713197

RESUMO

The self-assembly of short aromatic peptides and peptide derivatives into a variety of different nano- and microstructures (fibrillar gels, crystals, spheres, plates) is a promising route toward the creation of bio-compatible materials with often unexpected and useful properties. Furthermore, such simple self-assembling systems have been proposed as model systems for the self-assembly of longer peptides, a process that can be linked to biological function and malfunction. Much effort has been made in the last 15 years to explore the space of peptide sequences, chemical modifications and solvent conditions in order to maximise the diversity of assembly morphologies and properties. However, quantitative studies of the corresponding mechanisms of, and driving forces for, peptide self-assembly have remained relatively scarce until recently. In this chapter we review the current state of understanding of the thermodynamic driving forces and self-assembly mechanisms of short aromatic peptides into supramolecular structures. We will focus on experimental studies of the assembly process and our perspective will be centered around diphenylalanine (FF), a key motif of the amyloid ß sequence and a paradigmatic self-assembly building block. Our main focus is the basic physical chemistry and key structural aspects of such systems, and we will also compare the mechanism of dipeptide aggregation with that of longer peptide sequences into amyloid fibrils, with discussion on how these mechanisms may be revealed through detailed analysis of growth kinetics, thermodynamics and other fundamental properties of the aggregation process.


Assuntos
Peptídeos beta-Amiloides , Fenômenos Biofísicos , Termodinâmica , Peptídeos beta-Amiloides/química , Peptídeos beta-Amiloides/metabolismo , Dipeptídeos/química , Cinética
20.
Int J Mol Sci ; 20(19)2019 Oct 02.
Artigo em Inglês | MEDLINE | ID: mdl-31581677

RESUMO

The stiffness of the myosin cross-bridges is a key factor in analysing possible scenarios to explain myosin head changes during force generation in active muscles. The seminal study of Huxley and Simmons (1971: Nature 233: 533) suggested that most of the observed half-sarcomere instantaneous compliance (=1/stiffness) resides in the myosin heads. They showed with a so-called T1 plot that, after a very fast release, the half-sarcomere tension reduced to zero after a step size of about 60Å (later with improved experiments reduced to 40Å). However, later X-ray diffraction studies showed that myosin and actin filaments themselves stretch slightly under tension, which means that most (at least two-thirds) of the half sarcomere compliance comes from the filaments and not from cross-bridges. Here we have used a different approach, namely to model the compliances in a virtual half sarcomere structure in silico. We confirm that the T1 curve comes almost entirely from length changes in the myosin and actin filaments, because the calculated cross-bridge stiffness (probably greater than 0.4 pN/Å) is higher than previous studies have suggested. Our model demonstrates that the formulations produced by previous authors give very similar results to our model if the same starting parameters are used. However, we find that it is necessary to model the X-ray diffraction data as well as mechanics data to get a reliable estimate of the cross-bridge stiffness. In the light of the high cross-bridge stiffness found in the present study, we present a plausible modified scenario to describe aspects of the myosin cross-bridge cycle in active muscle. In particular, we suggest that, apart from the filament compliances, most of the cross-bridge contribution to the instantaneous T1 response may come from weakly-bound myosin heads, not myosin heads in strongly attached states. The strongly attached heads would still contribute to the T1 curve, but only in a very minor way, with a stiffness that we postulate could be around 0.1 pN/Å, a value which would generate a working stroke close to 100 Å from the hydrolysis of one ATP molecule. The new model can serve as a tool to calculate sarcomere elastic properties for any vertebrate striated muscle once various parameters have been determined (e.g., tension, T1 intercept, temperature, X-ray diffraction spacing results).


Assuntos
Modelos Moleculares , Contração Muscular , Músculo Esquelético/fisiologia , Miosinas/metabolismo , Fenômenos Biofísicos
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