RESUMEN
Amyloid proteins, which aggregate to form highly ordered structures, play a crucial role in various disease pathologies. Despite many previous studies on amyloid fibrils, which are an end product of protein aggregation, the structural characteristics of amyloid proteins in the early stage of aggregation and their related aggregation mechanism still remain elusive. The role of the amino acid sequence in the aggregation-prone structures of amyloid proteins at such a stage is not understood. Here, we have studied the sequence-dependent structural characteristics of islet amyloid polypeptide based on atomistic simulations and spectroscopic experiments. We show that the amino acid sequence determines non-bonded interactions that play a leading role in the formation of aggregation-prone conformations. Specifically, a single point mutation critically changes the population of aggregation-prone conformations, resulting in a change of the aggregation mechanism. Our simulation results were supported by experimental results suggesting that mutation affects the kinetics of aggregation and the structural characteristics of amyloid aggregates. Our study provides an insight into the role of sequence-dependent aggregation-prone conformations in the underlying mechanisms of amyloid aggregation.
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Polipéptido Amiloide de los Islotes Pancreáticos/química , Simulación de Dinámica Molecular , Humanos , Agregado de Proteínas , Conformación ProteicaRESUMEN
BACKGROUND: The objective of this study is to understand the relationship between physique, physical fitness, and balance performance for snowboard athletes. METHODS: We considered all Korean national snowboard athletes (n = 9 with 6 males and 3 females, age = 17.44 ± 4.42), who have an experience of competition at continental cup level, to measure their physique, physical fitness, and (both static and dynamic) balance. Static balance was evaluated based on one-legged standing, while dynamic balance was estimated using a stability platform. RESULTS: Static balance is strongly correlated (p < 0.05) with circumference of the left lower leg (34.49 ± 2.42 cm; ρ = 0.68), sit-up (57.56 ± 8.8; ρ = 0.72), sargent jump (50.22 ± 11.78 cm; ρ = 0.67), strength of bench press (39.11 ± 17.73 kg; ρ = 0.67), angle of left ankle dorsiflexion (73.78 ± 7.86°; ρ = 0.77), average extension strength at 180° for left knee (321 ± 63.95 %BW; ρ = 0.77) and right knee (337 ± 60.32 %BW; ρ = 0.77), and right knee peak flexion strength at 60° (148 ± 25.61 %BW; ρ = 0.73). Center dynamic balance is negatively correlated with circumference of the right lower leg (34.63 ± 2.38 cm; ρ = -0.67, p < 0.05), while right dynamic balance is positively correlated with left ankle flexion (148.44 ± 5.20°; ρ = 0.78, p < 0.05). CONCLUSION: Static balance is related to core muscle endurance and power, ankle flexibility, and knee stability, while dynamic balance is negatively correlated with circumference of the most frequently used lower leg (i.e., the leg dominating the snowboarding stance). The relationship between physique, physical fitness, and balance provides an insight into improving the balance performance of elite snowboard athletes through a training program that can affect the physique and physical fitness factors related to balance.
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Amyloid fibrils have recently been highlighted due to their excellent mechanical properties, which not only play a role in their biological functions but also imply their applications in biomimetic material design. Despite recent efforts to unveil how the excellent mechanical properties of amyloid fibrils originate, it has remained elusive how the anisotropic nanomechanical properties of hierarchically structured amyloid fibrils are determined. Here, we characterize the anisotropic nanomechanical properties of hierarchically structured amyloid fibrils using atomic force microscopy experiments and atomistic simulations. It is shown that the hierarchical structure of amyloid fibrils plays a crucial role in determining their radial elastic property but does not make any effect on their bending elastic property. This is attributed to the role of intermolecular force acting between the filaments (constituting the fibril) on the radial elastic modulus of amyloid fibrils. Our finding illustrates how the hierarchical structure of amyloid fibrils encodes their anisotropic nanomechanical properties. Our study provides key design principles of amyloid fibrils, which endow valuable insight into the underlying mechanisms of amyloid mechanics.
Asunto(s)
Amiloide/química , Fenómenos Mecánicos , Nanopartículas/química , Simulación por Computador , Elasticidad , Microscopía de Fuerza AtómicaRESUMEN
Amyloid ß (Aß) aggregates, which are a hallmark for neurodegenerative disease, are formed through a self-assembly process such as aggregation of Aß peptide chains. This aggregation process depends on the solvent conditions under which the proteins are aggregated. Nevertheless, the underlying mechanism of the ionic effect on the formation and stability of amyloid aggregates has not been fully understood. Here, we report how metal ions play a role in the formation and stability of Aß aggregates at different length scales, i.e. oligomers and fibrils. It is shown that the metal (i.e. zinc or copper) ion increases the stability of Aß oligomers, whereas the metal ion reduces the stability of Aß fibrils. In addition, we found that zinc ions are able to more effectively destabilize fibril structures than copper ions. Metal ion-mediated (de)stabilization of Aß oligomers (or fibrils) is attributed to the critical effect of the metal ion on the ß-sheet rich crystalline structure of the amyloid aggregate and the status of hydrogen bonds within the aggregate. Our study sheds light on the role of the metal ion in stabilizing the amyloid oligomers known as a toxic agent (to functional cells), which is consistent with clinical observation that high concentrations of metal ions are found in patients suffering from neurodegenerative diseases.
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Péptidos beta-Amiloides/química , Iones/química , Metales/química , Péptidos beta-Amiloides/metabolismo , Cristalización , Estabilidad ProteicaRESUMEN
Amyloid fibrils are responsible for pathogenesis of various diseases and exhibit the structural feature of an ordered, hierarchical structure such as multi-stranded helical structure. As the multi-strandedness of amyloid fibrils has recently been found to be highly correlated with their toxicity and infectivity, it is necessary to study how the hierarchical (i.e. multi-stranded) structure of amyloid fibril is formed. Moreover, although it has recently been reported that the nanomechanics of amyloid proteins plays a key role on the amyloid-induced pathogenesis, a critical role that the multi-stranded helical structure of the fibrils plays in their nanomechanical properties has not fully characterized. In this work, we characterize the morphology and mechanical properties of multi-stranded amyloid fibrils by using equilibrium molecular dynamics simulation and elastic network model. It is shown that the helical pitch of multi-stranded amyloid fibril is linearly proportional to the number of filaments comprising the amyloid fibril, and that multi-strandedness gives rise to improving the bending rigidity of the fibril. Moreover, we have also studied the morphology and mechanical properties of a single protofilament (filament) in order to understand the effect of cross-ß structure and mutation on the structures and mechanical properties of amyloid fibrils. Our study sheds light on the underlying design principles showing how the multi-stranded amyloid fibril is formed and how the structure of amyloid fibrils governs their nanomechanical properties.
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Amiloide/química , Simulación de Dinámica Molecular , Fenómenos BiomecánicosRESUMEN
Amyloid fibrils have recently received attention due to their remarkable mechanical properties, which are highly correlated with their biological functions. We have studied the mechanical deformation mechanisms and properties of amyloid fibrils as a function of their length scales by using atomistic simulations. It is shown that the length of amyloid fibrils plays a role in their deformation and fracture mechanisms in such a way that the competition between shear and bending deformations is highly dependent on the fibril length, and that as the fibril length increases, so does the bending strength of the fibril while its shear strength decreases. The dependence of rupture force for amyloid fibrils on their length is elucidated using the Bell model, which suggests that the rupture force of the fibril is determined from the hydrogen bond rupture mechanism that critically depends on the fibril length. We have measured the toughness of amyloid fibrils, which is shown to depend on the fibril length. In particular, the toughness of the fibril with its length of â¼3 nm is estimated to be â¼30 kcal mol(-1) nm(-3), comparable to that of a spider silk crystal with its length of â¼2 nm. Moreover, we have shown the important effect of the pulling rate on the mechanical deformation mechanisms and properties of amyloid fibril. It is found that as the pulling rate increases, so does the contribution of the shear effect to the elastic deformation of the amyloid fibril with its length of <10 nm. However, we found that the deformation mechanism of the amyloid fibril with its length of >15 nm is almost independent of the pulling rate. Our study sheds light on the role of the length scale of amyloid fibrils and the pulling rate in their mechanical behaviors and properties, which may provide insights into how the excellent mechanical properties of protein fibrils can be determined.
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Amiloide/química , Fenómenos Mecánicos , Simulación de Dinámica Molecular , Fenómenos Biomecánicos , Elasticidad , Estructura Secundaria de ProteínaRESUMEN
Single molecule experiments and simulations have been widely used to characterize the unfolding and folding pathways of different proteins. However, with few exceptions, these tools have not been applied to study prion protein, PrP(C), whose misfolded form PrP(Sc) can induce a group of fatal neurodegenerative diseases. Here, we apply novel atomistic modeling based on potential energy surface exploration to study the constant force unfolding of human PrP at time scales inaccessible with standard molecular dynamics. We demonstrate for forces around 100 pN, prion forms a stable, three-stranded ß-sheet-like intermediate configuration containing residues 155-214 with a lifetime exceeding hundreds of nanoseconds. A mutant without the disulfide bridge shows lower stability during the unfolding process but still forms the three-stranded structure. The simulations thus not only show the atomistic details of the mechanically induced structural conversion from the native α-helical structure to the ß-rich-like form but also lend support to the structural theory that there is a core of the recombinant PrP amyloid, a misfolded form reported to induce transmissible disease, mapping to C-terminal residues ≈160-220.
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Priones/química , Desplegamiento Proteico , Humanos , Enlace de Hidrógeno , Simulación de Dinámica Molecular , Mutación , Priones/genética , Estabilidad Proteica , Estructura Secundaria de ProteínaRESUMEN
Proteins with multiple domains play pivotal roles in various biological processes, necessitating a thorough understanding of their structural stability and functional interplay. Here, a structure-guided protein engineering approach is proposed to develop thermostable Cas9 (CRISPR-associated protein 9) variant for CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats) interference applications. By employing thermodynamic analysis, combining distance mapping and molecular dynamics simulations, deletable domains are identified to enhance stability while preserving the DNA recognition function of Cas9. The resulting engineered Cas9, termed small and dead form Cas9, exhibits improved thermostability and maintains target DNA recognition function. Cryo-electron microscopy analysis reveals structural integrity with reduced atomic density in the deleted domain. Fusion with functional elements enables intracellular delivery and nuclear localization, demonstrating efficient gene suppression in diverse cell types. Direct delivery in the mouse brain shows enhanced knockdown efficiency, highlighting the potential of structure-guided engineering to develop functional CRISPR systems tailored for specific applications. This study underscores the significance of integrating computational and experimental approaches for protein engineering, offering insights into designing tailored molecular tools for precise biological interventions.
RESUMEN
The small organic molecules, known as osmolytes being ubiquitously present in different cell types, affect protein folding, stability and aggregation. However, it is unknown how the osmolytes affect the nanomechanical unfolding behavior of protein domain. Here, we show the osmolyte-dependent mechanical unfolding properties of protein titin immunoglobulin-27 (I27) domain using an atomic force microscopy (AFM)-based single-molecule force spectroscopy. We found that amines and methylamines improved the mechanical stability of I27 domain, whereas polyols had no effect. Interestingly, glycine betaine (GB) or trimethylamine-N-oxide (TMAO) increased the average unfolding force of the protein domain. The kinetic parameters analyzed at single-molecule level reveal that stabilizing effect of osmolytes is due to a decrease in the unfolding rate constant of I27, which was confirmed by molecular dynamics simulations. Our study reveals different effects that diverse osmolytes have on the mechanical properties of the protein, and suggests the potential use of osmolytes in modulating the mechanical stability of proteins required for various nano-biotechnological applications.
Asunto(s)
Pliegue de Proteína , Proteínas , Dominios Proteicos , Proteínas/química , Simulación de Dinámica Molecular , Fenómenos Mecánicos , Inmunoglobulinas/química , Desplegamiento Proteico , Microscopía de Fuerza Atómica/métodosRESUMEN
Single-molecule mechanical manipulation has enabled quantitative understanding of not only the kinetics of both bond rupture and protein unfolding, but also the free energy landscape of chemical bond and/or protein folding. Despite recent studies reporting the role of loading device in bond rupture, a loading device effect on protein unfolding mechanics has not been well studied. In this work, we have studied the effect of loading-device stiffness on the kinetics of both bond rupture and protein unfolding mechanics using Brownian dynamics simulations. It is shown that bond rupture forces are dependent on not only loading rate but also the stiffness of loading device, and that protein unfolding mechanics is highly correlated with the stiffness of loading device. Our study sheds light on the importance of loading device effect on the mechanically induced bond ruptures and protein unfolding.
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Fenómenos Mecánicos , Simulación de Dinámica Molecular , Desplegamiento Proteico , Ubiquitina/química , Fenómenos Biomecánicos , Movimiento , Estructura Secundaria de Proteína , Ubiquitina/metabolismoRESUMEN
Normal mode analysis (NMA) with coarse-grained model, such as elastic network model (ENM), has allowed the quantitative understanding of protein dynamics. As the protein size is increased, there emerges the expensive computational process to find the dynamically important low-frequency normal modes due to diagonalization of massive Hessian matrix. In this study, we have provided the domain decomposition-based structural condensation method that enables the efficient computations on low-frequency motions. Specifically, our coarse-graining method is established by coupling between model condensation (MC; Eom et al., J Comput Chem 2007, 28, 1400) and component mode synthesis (Kim et al., J Chem Theor Comput 2009, 5, 1931). A protein structure is first decomposed into substructural units, and then each substructural unit is coarse-grained by MC. Once the NMA is implemented to coarse-grained substructural units, normal modes and natural frequencies for each coarse-grained substructural unit are assembled by using geometric constraints to provide the normal modes and natural frequencies for whole protein structure. It is shown that our coarse-graining method enhances the computational efficiency for analysis of large protein complexes. It is clearly suggested that our coarse-graining method provides the B-factors of 100 large proteins, quantitatively comparable with those obtained from original NMA, with computational efficiency. Moreover, the collective behaviors and/or the correlated motions for model proteins are well delineated by our suggested coarse-grained models, quantitatively comparable with those computed from original NMA. It is implied that our coarse-grained method enables the computationally efficient studies on conformational dynamics of large protein complex.
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Conformación Proteica , Proteínas/química , Modelos MolecularesRESUMEN
Nanomechanical resonators have recently been highlighted because of their remarkable ability to perform both sensing and detection. Since the nanomechanical resonators are characterized by a large surface-to-volume ratio, it is implied that the surface effect plays a substantial role on not only the resonance but also the sensing performance of nanomechanical resonators. In this work, we have studied the role of surface effect on the detection sensitivity of a nanoresonator that undergoes either harmonic vibration or nonlinear oscillation based on the continuum elastic model such as an elastic beam model. It is shown that the surface effect makes an impact on both harmonic resonance and nonlinear oscillations, and that the sensing performance is dependent on the surface effect. Moreover, we have also investigated the surface effect on the mechanical tuning of resonance and sensing performance. It is interestingly found that the mechanical tuning of resonance is independent of the surface effect, while the mechanical tuning of sensing performance is determined by the surface effect. Our study sheds light on the importance of the surface effect on the sensing performance of nanoresonators.
RESUMEN
Self-aggregation of amyloid-ß (Aß) peptides has been known to play a vital role in the onset stage of neurodegenerative diseases, indicating the necessity of understanding the aggregation process of Aß peptides. Despite previous studies on the aggregation process of Aß peptides, the aggregation pathways of Aß isoforms (i.e., Aß40 and Aß42) and their related structures have not been fully understood yet. Here, we study the aggregation pathways of Aß40 and Aß42, and the structures of Aß40 and Aß42 aggregates during the process, based on fluorescence and atomic force microscopy (AFM) experiments. It is shown that in the beginning of aggregation process for both Aß40 and Aß42, a number of particles (i.e., spherical oligomers) are formed. These particles are subsequently self-assembled together, resulting in the formation of different shapes of amyloid fibrils. Our finding suggests that the different aggregation pathways of Aß isoforms lead to the amyloid fibrils with contrasting structure.
Asunto(s)
Péptidos beta-Amiloides/química , Amiloide/química , Fragmentos de Péptidos/química , Péptidos/química , Enfermedad de Alzheimer/metabolismo , Benzotiazoles/química , Humanos , Microscopía de Fuerza Atómica , Microscopía Fluorescente , Enfermedades Neurodegenerativas/metabolismo , Unión Proteica , Conformación Proteica , Isoformas de ProteínasRESUMEN
Communicated by Ramaswamy H. Sarma.
Asunto(s)
Conectina , Fenómenos Biofísicos , Conectina/genética , Conectina/metabolismo , Ligandos , Dominios ProteicosRESUMEN
Investigation of the dielectric properties of cell membranes plays an important role in understanding the biological activities that sustain cellular life and realize cellular functionalities. Herein, the variable dielectric polarization characteristics of cell membranes are reported. In controlling the dielectric polarization of a cell using dielectrophoresis force spectroscopy, different cellular crossover frequencies were observed by modulating both the direction and sweep rate of the frequency. The crossover frequencies were used for the extraction of the variable capacitance, which is involved in the dielectric polarization across the cell membranes. In addition, this variable phenomenon was investigated by examining cells whose membranes were cholesterol-depleted with methyl-ß-cyclodextrin, which verified a strong correlation between the variable dielectric polarization characteristics and membrane composition changes. This study presented the dielectric polarization properties in live cells' membranes that can be modified by the regulation of external stimuli and provided a powerful platform to explore cellular membrane dielectric polarization.
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Membrana Celular/metabolismo , Membrana Celular/efectos de los fármacos , Supervivencia Celular , Impedancia Eléctrica , Humanos , Células MCF-7 , beta-Ciclodextrinas/farmacologíaRESUMEN
Mechanical characterization of protein molecules has played a role on gaining insight into the biological functions of proteins, because some proteins perform the mechanical function. Here, we present the mesoscopic model of biological protein materials composed of protein crystals prescribed by Go potential for characterization of elastic behavior of protein materials. Specifically, we consider the representative volume element (RVE) containing the protein crystals represented by C(alpha) atoms, prescribed by Go potential, with application of constant normal strain to RVE. The stress-strain relationship computed from virial stress theory provides the nonlinear elastic behavior of protein materials and their mechanical properties such as Young's modulus, quantitatively and/or qualitatively comparable with mechanical properties of biological protein materials obtained from experiments and/or atomistic simulations. Further, we discuss the role of native topology on the mechanical properties of protein crystals. It is shown that parallel strands (hydrogen bonds in parallel) enhance the mechanical resilience of protein materials.
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Proteínas/química , Estrés Mecánico , Simulación por Computador , Cristalografía por Rayos X , Elasticidad , Modelos Moleculares , Conformación ProteicaRESUMEN
Protein dynamics is essential for gaining insight into biological functions of proteins. Although protein dynamics is well delineated by molecular model, the molecular model is computationally prohibited for simulating large protein structures. In this work, we provide a multiscale network model (MNM) that allows the efficient computation on low-frequency normal modes related to structural deformation of proteins as well as dynamic behavior of functional sites. Specifically, MNM consists of two regions, one of which is described as a low-resolution structure, while the other is dictated by a high-resolution structure. The high-resolution regions using all alpha carbons of the protein are mainly binding site parts, which play a critical function in molecules, while the low-resolution parts are constructed from a further coarse-grained model (not using all alpha carbons). The feasibility of MNM to observe the cooperative motion of a protein structure was validated. It was shown that the MNM enables us to understand functional motion of proteins with computational efficiency.
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Modelos Moleculares , Proteínas/química , Proteínas/metabolismo , Movimiento , Miosinas/química , Miosinas/metabolismo , Inhibidores de Proteasas/química , Inhibidores de Proteasas/metabolismo , Conformación Proteica , TemperaturaRESUMEN
Quantitative understanding of the mechanical behavior of biological liquid crystals such as proteins is essential for gaining insight into their biological functions, since some proteins perform notable mechanical functions. Recently, single-molecule experiments have allowed not only the quantitative characterization of the mechanical behavior of proteins such as protein unfolding mechanics, but also the exploration of the free energy landscape for protein folding. In this work, we have reviewed the current state-of-art in single-molecule bioassays that enable quantitative studies on protein unfolding mechanics and/or various molecular interactions. Specifically, single-molecule pulling experiments based on atomic force microscopy (AFM) have been overviewed. In addition, the computational simulations on single-molecule pulling experiments have been reviewed. We have also reviewed the AFM cantilever-based bioassay that provides insight into various molecular interactions. Our review highlights the AFM-based single-molecule bioassay for quantitative characterization of biological liquid crystals such as proteins.
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Cristales Líquidos/química , Modelos Químicos , Simulación de Dinámica Molecular , Fenómenos Biomecánicos , Enlace de Hidrógeno , Microscopía de Fuerza Atómica , Desplegamiento Proteico , Proteínas/química , TermodinámicaRESUMEN
Atomic force microscopy (AFM) has recently attracted much attention due to its ability to analyze biomolecular interactions and to detect certain biomolecules, which play a crucial role in disease expression. Despite recent studies reporting AFM imaging for the analyses of biomolecules, the application of AFM-based cancer-specific biomolecule/cell detection has remained largely underexplored, especially for the early diagnosis of cancer. In this paper, we review the recent attempts, including our efforts, to analyze and detect cancer-specific biomolecules and cancer cells. We particularly focus on two AFM-based cancer diagnosis techniques: (i) AFM imaging-based biomolecular and cellular detection, (ii) AFM cantilever-based biomolecular sensing and cell analysis. It is shown that AFM-based biomolecular detection has been applied for not only early diagnosing cancer, by measuring the minute amount of cancer-specific proteins, but also monitoring of cancer progression, by correlating the amount of cancer-specific proteins with the progression of cancer. In addition, AFM-based cell imaging and detection have been employed for diagnosing cancer, by detecting cancerous cells in tissue, as well as understanding cancer progression, by characterizing the dynamics of cancer cells. This review, therefore, highlights AFM-based biomolecule/cell detection, which will pave the way for developing a fast and point-of-care diagnostic system for biomedical applications.
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Microscopía de Fuerza Atómica/métodos , Neoplasias/diagnóstico , Animales , HumanosRESUMEN
We report the nanomechanical microcantilevers operated in vibration modes (oscillation) with use of RNA aptamers as receptor molecules for label-free detection of hepatitis C virus (HCV) helicase. The nanomechanical detection principle is that the ligand-receptor binding on the microcantilever surface induces the dynamic response change of microcantilevers. We implemented the label-free detection of HCV helicase in the low concentration as much as 100 pg/ml from measuring the dynamic response change of microcantilevers. Moreover, from the recent studies showing that the ligand-receptor binding generates the surface stress on the microcantilever, we estimate the surface stress, on the oscillating microcantilevers, induced by ligand-receptor binding, i.e. binding between HCV helicase and RNA aptamer. In this article, it is suggested that the oscillating microcantilevers with use of RNA aptamers as receptor molecules may enable one to implement the sensitive label-free detection of very small amount of small-scale proteins.