RESUMO
The nuclear pore complex regulates nucleocytoplasmic transport by means of a tightly synchronized suite of biochemical reactions. The physicochemical properties of the translocating cargos are emerging as master regulators of their shuttling dynamics. As well as being affected by molecular weight and surface-exposed amino acids, the kinetics of the nuclear translocation of protein cargos also depend on their nanomechanical properties, yet the mechanisms underpinning the mechanoselectivity of the nuclear pore complex are unclear. Here we show that proteins with locally soft regions in the vicinity of the nuclear-localization sequence exhibit higher nuclear-import rates, and that such mechanoselectivity is specifically impaired upon knocking down nucleoporin 153, a key protein in the nuclear pore complex. This allows us to design a short, easy-to-express and chemically inert unstructured peptide tag that accelerates the nuclear-import rate of stiff protein cargos. We also show that U2OS osteosarcoma cells expressing the peptide-tagged myocardin-related transcription factor import this mechanosensitive protein to the nucleus at higher rates and display faster motility. Locally unstructured regions lower the free-energy barrier of protein translocation and might offer a control mechanism for nuclear mechanotransduction.
RESUMO
The reversible unfolding and refolding of proteins is a regulatory mechanism of tissue elasticity and signalling used by cells to sense and adapt to extracellular and intracellular mechanical forces. However, most of these proteins exhibit low mechanical stability, posing technical challenges to the characterization of their conformational dynamics under force. Here, we detail step-by-step instructions for conducting single-protein nanomechanical experiments using ultra-stable magnetic tweezers, which enable the measurement of the equilibrium conformational dynamics of single proteins under physiologically relevant low forces applied over biologically relevant timescales. We report the basic principles determining the functioning of the magnetic tweezer instrument, review the protein design strategy and the fluid chamber preparation and detail the procedure to acquire and analyze the unfolding and refolding trajectories of individual proteins under force. This technique adds to the toolbox of single-molecule nanomechanical techniques and will be of particular interest to those interested in proteins involved in mechanosensing and mechanotransduction. The procedure takes 4 d to complete, plus an additional 6 d for protein cloning and production, requiring basic expertise in molecular biology, surface chemistry and data analysis.
Assuntos
Proteínas , Proteínas/química , Proteínas/metabolismo , Imagem Individual de Molécula/métodos , Magnetismo/métodos , Nanotecnologia/métodos , Conformação Proteica , Dobramento de ProteínaRESUMO
Mechanical forces are critical to protein function across many biological contexts-from bacterial adhesion to muscle mechanics and mechanotransduction processes. Hence, understanding how mechanical forces govern protein activity has developed into a central scientific question. In this context, single-molecule magnetic tweezers has recently emerged as a valuable experimental tool, offering the capability to measure single proteins over physiologically relevant forces and timescales. In this chapter, we present a detailed protocol for the assembly and operation of our magnetic tape head tweezers instrument, specifically tailored to investigate protein dynamics. Our instrument boasts a simplified microscope design and incorporates a magnetic tape head as the force-generating apparatus, facilitating precise force control and enhancing its temporal stability, enabling the study of single protein mechanics over extended timescales spanning several hours or even days. Moreover, its straightforward and cost-effective design ensures its accessibility to the wider scientific community. We anticipate that this technique will attract widespread interest within the growing field of mechanobiology and expect that this chapter will provide facilitated accessibility to this technology.
Assuntos
Fenômenos Mecânicos , Mecanotransdução Celular , Proteínas , Magnetismo/métodos , Fenômenos Magnéticos , Pinças ÓpticasRESUMO
The classical "one sequence, one structure, one function" paradigm has shaped much of our intuition of how proteins work inside the cell. Partially due to the insight provided by bulk biochemical assays, individual biomolecules are often assumed to behave as identical entities, and their characterization relies on ensemble averages that flatten any conformational diversity into a unique phenotype. While the emergence of single-molecule techniques opened the gates to interrogating individual molecules, technical shortcomings typically limit the duration of these measurements, which precludes a complete characterization of an individual protein and, hence, capturing the heterogeneity among molecular populations. Here, we introduce an ultrastable magnetic tweezers design, which enables us to measure the folding dynamics of a single protein during several uninterrupted days with high temporal and spatial resolution. Thanks to this instrumental development, we fully characterize the nanomechanics of two proteins with a very distinct force response, the talin R3IVVI domain and protein L. Days-long recordings on the same protein individual accumulate thousands of folding transitions with submicrosecond resolution, allowing us to reconstruct their free energy landscapes and describe how they evolve with force. By mapping the nanomechanical identity of many different protein individuals, we directly capture their molecular diversity as a quantifiable dispersion on their force response and folding kinetics. By significantly expanding the measurable timescales, our instrumental development offers a tool for profiling individual molecules, opening the gates to directly characterizing biomolecular heterogeneity.
Assuntos
Dobramento de Proteína , Proteínas , Humanos , Proteínas/química , Fenômenos Mecânicos , Cinética , Conformação MolecularRESUMO
During organ formation, progenitor cells need to acquire different cell identities and organize themselves into distinct structural units. How these processes are coordinated and how tissue architecture(s) is preserved despite the dramatic cell rearrangements occurring in developing organs remain unclear. Here, we identified cellular rearrangements between acinar and ductal progenitors as a mechanism to drive branching morphogenesis in the pancreas while preserving the integrity of the acinar-ductal functional unit. Using ex vivo and in vivo mouse models, we found that pancreatic ductal cells form clefts by protruding and pulling on the acinar basement membrane, which leads to acini splitting. Newly formed acini remain connected to the bifurcated branches generated by ductal cell rearrangement. Insulin growth factor (IGF)/phosphatidylinositol 3-kinase (PI3K) pathway finely regulates this process by controlling pancreatic ductal tissue fluidity, with a simultaneous impact on branching and cell fate acquisition. Together, our results explain how acinar structure multiplication and branch bifurcation are synchronized during pancreas organogenesis.
Assuntos
Fosfatidilinositol 3-Quinase , Fosfatidilinositol 3-Quinases , Camundongos , Animais , Fosfatidilinositol 3-Quinases/metabolismo , Fosfatidilinositol 3-Quinase/metabolismo , Pâncreas , Células Acinares/metabolismo , Morfogênese/fisiologia , Peptídeos e Proteínas de Sinalização Intercelular/metabolismoRESUMO
Dedifferentiation is the process by which terminally differentiated cells acquire the properties of stem cells. During mouse skin wound healing, the differentiated Gata6-lineage positive cells of the sebaceous duct are able to dedifferentiate. Here we have integrated lineage tracing and single-cell mRNA sequencing to uncover the underlying mechanism. Gata6-lineage positive and negative epidermal stem cells in wounds are transcriptionally indistinguishable. Furthermore, in contrast to reprogramming of induced pluripotent stem cells, the same genes are expressed in the epidermal dedifferentiation and differentiation trajectories, indicating that dedifferentiation does not involve adoption of a new cell state. We demonstrate that dedifferentiation is not only induced by wounding, but also by retinoic acid treatment or mechanical expansion of the epidermis. In all three cases, dedifferentiation is dependent on the master transcription factor c-Myc. Mechanotransduction and actin-cytoskeleton remodelling are key features of dedifferentiation. Our study elucidates the molecular basis of epidermal dedifferentiation, which may be generally applicable to adult tissues.
Assuntos
Desdiferenciação Celular , Mecanotransdução Celular , Animais , Camundongos , Desdiferenciação Celular/genética , Diferenciação Celular , Células Epidérmicas , EpidermeRESUMO
The talin-vinculin axis is a key mechanosensing component of cellular focal adhesions. How talin and vinculin respond to forces and regulate one another remains unclear. By combining single-molecule magnetic tweezers experiments, Molecular Dynamics simulations, actin-bundling assays, and adhesion assembly experiments in live cells, we here describe a two-ways allosteric network within vinculin as a regulator of the talin-vinculin interaction. We directly observe a maturation process of vinculin upon talin binding, which reinforces the binding to talin at a rate of 0.03 s-1. This allosteric transition can compete with force-induced dissociation of vinculin from talin only at forces up to 10 pN. Mimicking the allosteric activation by mutation yields a vinculin molecule that bundles actin and localizes to focal adhesions in a force-independent manner. Hence, the allosteric switch confines talin-vinculin interactions and focal adhesion build-up to intermediate force levels. The 'allosteric vinculin mutant' is a valuable molecular tool to further dissect the mechanical and biochemical signalling circuits at focal adhesions and elsewhere.
Assuntos
Actinas , Talina , Actinas/metabolismo , Talina/metabolismo , Vinculina/genética , Vinculina/metabolismo , Regulação Alostérica , Adesões Focais/metabolismo , Ligação ProteicaRESUMO
Statistical mechanics can describe the major conformational ensembles determining the equilibrium free-energy landscape of a folding protein. The challenge is to capture the full repertoire of low-occurrence conformations separated by high kinetic barriers that define complex landscapes. Computationally, enhanced sampling methods accelerate the exploration of molecular rare events. However, accessing the entire protein's conformational space in equilibrium experiments requires technological developments to enable extended observation times. We developed single-molecule magnetic tweezers to capture over a million individual transitions as a single talin protein unfolds and refolds under force in equilibrium. When observed at classically-probed timescales, talin folds in an apparently uncomplicated two-state manner. As the sampling time extends from minutes to days, the underlying energy landscape exhibits gradually larger signatures of complexity, involving a finite number of well-defined rare conformations. A fluctuation analysis allows us to propose plausible structures of each low-probability conformational state. The physiological relevance of each distinct conformation can be connected to the binding of the cytoskeletal protein vinculin, suggesting an extra layer of complexity in talin-mediated mechanotransduction. More generally, our experiments directly test the fundamental notion that equilibrium dynamics depend on the observation timescale.
RESUMO
Gram-positive bacteria colonize mucosal tissues, withstanding large mechanical perturbations such as coughing, which generate shear forces that exceed the ability of non-covalent bonds to remain attached. To overcome these challenges, the pathogen Streptococcus pyogenes utilizes the protein Cpa, a pilus tip-end adhesin equipped with a Cys-Gln thioester bond. The reactivity of this bond towards host surface ligands enables covalent anchoring; however, colonization also requires cell migration and spreading over surfaces. The molecular mechanisms underlying these seemingly incompatible requirements remain unknown. Here we demonstrate a magnetic tweezers force spectroscopy assay that resolves the dynamics of the Cpa thioester bond under force. When folded at forces <6 pN, the Cpa thioester bond reacts reversibly with amine ligands, which are common in inflammation sites; however, mechanical unfolding and exposure to forces >6 pN block thioester reformation. We hypothesize that this folding-coupled reactivity switch (termed a smart covalent bond) could allow the adhesin to undergo binding and unbinding to surface ligands under low force and remain covalently attached under mechanical stress.
Assuntos
Adesinas Bacterianas/química , Fímbrias Bacterianas/química , Adesinas Bacterianas/análise , Adesinas Bacterianas/metabolismo , Fímbrias Bacterianas/metabolismo , Ligação Proteica , Dobramento de Proteína , Streptococcus pyogenes/química , Streptococcus pyogenes/metabolismoRESUMO
Cells continually sample their mechanical environment using exquisite force sensors such as talin, whose folding status triggers mechanotransduction pathways by recruiting binding partners. Mechanical signals in biology change quickly over time and are often embedded in noise; however, the mechanics of force-sensing proteins have only been tested using simple force protocols, such as constant or ramped forces. Here, using our magnetic tape head tweezers design, we measure the folding dynamics of single talin proteins in response to external mechanical noise and cyclic force perturbations. Our experiments demonstrate that talin filters out external mechanical noise but detects periodic force signals over a finely tuned frequency range. Hence, talin operates as a mechanical band-pass filter, able to read and interpret frequency-dependent mechanical information through its folding dynamics. We describe our observations in the context of stochastic resonance, which we propose as a mechanism by which mechanosensing proteins could respond accurately to force signals in the naturally noisy biological environment.
Assuntos
Mecanotransdução Celular , Talina/fisiologia , Domínios Proteicos , Dobramento de Proteína , Imagem Individual de MoléculaRESUMO
Vinculin binds unfolded talin domains in focal adhesions, which recruits actin filaments to reinforce the mechanical coupling of this organelle. However, it remains unknown how this interaction is regulated and its impact on the force transmission properties of this mechanotransduction pathway. Here, we use magnetic tweezers to measure the interaction between vinculin head and the talin R3 domain under physiological forces. For the first time, we resolve individual binding events as a short contraction of the unfolded talin polypeptide caused by the reformation of the vinculin-binding site helices, which dictates a biphasic mechanism that regulates this interaction. Force favors vinculin binding by unfolding talin and exposing the vinculin-binding sites; however, the coil-to-helix contraction introduces an energy penalty that increases with force, defining an optimal binding regime. This mechanism implies that the talin-vinculin-actin association could operate as a negative feedback mechanism to stabilize force on focal adhesions.
RESUMO
Single-molecule methods using recombinant proteins have generated transformative hypotheses on how mechanical forces are generated and sensed in biological tissues. However, testing these mechanical hypotheses on proteins in their natural environment remains inaccesible to conventional tools. To address this limitation, here we demonstrate a mouse model carrying a HaloTag-TEV insertion in the protein titin, the main determinant of myocyte stiffness. Using our system, we specifically sever titin by digestion with TEV protease, and find that the response of muscle fibers to length changes requires mechanical transduction through titin's intact polypeptide chain. In addition, HaloTag-based covalent tethering enables examination of titin dynamics under force using magnetic tweezers. At pulling forces < 10 pN, titin domains are recruited to the unfolded state, and produce 41.5 zJ mechanical work during refolding. Insertion of the HaloTag-TEV cassette in mechanical proteins opens opportunities to explore the molecular basis of cellular force generation, mechanosensing and mechanotransduction.
Assuntos
Conectina/metabolismo , Endopeptidases/genética , Especificidade de Órgãos , Animais , Fenômenos Biomecânicos , Conectina/química , Feminino , Proteínas Imobilizadas/metabolismo , Magnetismo , Camundongos , Músculos/metabolismo , Músculos/ultraestrutura , Pinças Ópticas , Fenótipo , Dobramento de Proteína , Análise EspectralRESUMO
Force spectroscopy techniques are often used to learn about the free energy landscape of single biomolecules, typically by recovering free energy quantities that, extrapolated to zero force, are compared to those measured in bulk experiments. However, it is not always clear how the information obtained from a mechanically perturbed system can be related to the information obtained using other denaturants since tensioned molecules unfold and refold along a reaction coordinate imposed by the force, which is not likely to be meaningful in its absence. Here, we explore this dichotomy by investigating the unfolding landscape of a model protein, which is unfolded first mechanically through typical force spectroscopy-like protocols and next thermally. When unfolded by nonequilibrium force extension and constant force protocols, we recover a simple two-barrier landscape as the protein reaches the extended conformation through a metastable intermediate. Interestingly, folding-unfolding equilibrium simulations at low forces suggested a totally different scenario, where this metastable state plays little role in the unfolding mechanism, and the protein unfolds through two competing pathways [R. Tapia-Rojo et al., J. Chem. Phys. 141, 135102 (2014)]. Finally, we use Markov state models to describe the configurational space of the unperturbed protein close to the critical temperature. The thermal dynamics is well understood by a one-dimensional landscape along an appropriate reaction coordinate, however it is very different from the mechanical picture. In this sense, the results of our protein model for the mechanical and thermal descriptions provide incompatible views of the folding/unfolding landscape of the system, and the estimated quantities to zero force result are hard to interpret.
Assuntos
Desdobramento de Proteína , Proteínas/química , Temperatura , Cadeias de Markov , Fenômenos Mecânicos , Modelos Moleculares , Conformação ProteicaRESUMO
The delivery of mechanical power, a crucial component of animal motion, is constrained by the universal compromise between the force and the velocity of its constituent molecular systems. While the mechanisms of force generation have been studied at the single molecular motor level, there is little understanding of the magnitude of power that can be generated by folding proteins. Here, we use single-molecule force spectroscopy techniques to measure the force-velocity relation of folding titin domains that contain single internal disulfide bonds, a common feature throughout the titin I-band. We find that formation of the disulfide regulates the peak power output of protein folding in an all-or-none manner, providing at 6.0 pN, for example, a boost from 0 to 6,000 zW upon oxidation. This mechanism of power generation from protein folding is of great importance for muscle, where titin domains may unfold and refold with each extension and contraction of the sarcomere.
Assuntos
Conectina/química , Conectina/metabolismo , Dobramento de Proteína , Fenômenos Biomecânicos , Dissulfetos/metabolismo , Modelos Biológicos , Chaperonas Moleculares/metabolismo , Oxirredução , Oxirredutases/metabolismo , Peptídeos/metabolismo , Isomerases de Dissulfetos de Proteínas/metabolismo , Domínios ProteicosRESUMO
Magnetic tape heads are ubiquitously used to read and record on magnetic tapes in technologies as diverse as old VHS tapes, modern hard-drive disks, or magnetic bands on credit cards. Their design highlights the ability to convert electric signals into fluctuations of the magnetic field at very high frequencies, which is essential for the high-density storage demanded nowadays. Here, we twist this conventional use of tape heads to implement one in a magnetic tweezers design, which offers the unique capability of changing the force with a bandwidth of â¼10 kHz. We calibrate our instrument by developing an analytical expression that predicts the magnetic force acting on a superparamagnetic bead based on the Karlqvist approximation of the magnetic field created by a tape head. This theory is validated by measuring the force dependence of protein L unfolding/folding step sizes and the folding properties of the R3 talin domain. We demonstrate the potential of our instrument by carrying out millisecond-long quenches to capture the formation of the ephemeral molten globule state in protein L, which has never been observed before. Our instrument provides the capability of interrogating individual molecules under fast-changing forces with a control and resolution below a fraction of a piconewton, opening a range of force spectroscopy protocols to study protein dynamics under force.
Assuntos
Campos Magnéticos , Proteínas/química , Análise Espectral , Desenho de Equipamento , Fenômenos Mecânicos , Microscopia de Força Atômica , Dobramento de Proteína , Análise Espectral/instrumentação , Análise Espectral/métodosRESUMO
Bacteria anchor to their host cells through their adhesive pili, which must resist the large mechanical stresses induced by the host as it attempts to dislodge the pathogens. The pili of gram-positive bacteria are constructed as a single polypeptide made of hundreds of pilin repeats, which contain intramolecular isopeptide bonds strategically located in the structure to prevent their unfolding under force, protecting the pilus from degradation by extant proteases and oxygen radicals. Here, we demonstrate the design of a short peptide that blocks the formation of the isopeptide bond present in the pilin Spy0128 from the human pathogen Streptococcus pyogenes, resulting in mechanically labile pilin domains. We use a combination of protein engineering and atomic-force microscopy force spectroscopy to demonstrate that the peptide blocks the formation of the native isopeptide bond and compromises the mechanics of the domain. While an intact Spy0128 is inextensible at any force, peptide-modified Spy0128 pilins readily unfold at very low forces, marking the abrogation of the intramolecular isopeptide bond as well as the absence of a stable pilin fold. We propose that isopeptide-blocking peptides could be further developed as a type of highly specific antiadhesive antibiotics to treat gram-positive pathogens.
Assuntos
Antibacterianos/química , Proteínas de Fímbrias/antagonistas & inibidores , Proteínas de Fímbrias/química , Peptídeos/química , Dobramento de Proteína , Streptococcus pyogenes/química , Antibacterianos/farmacologia , Proteínas de Fímbrias/metabolismo , Humanos , Peptídeos/farmacologia , Domínios Proteicos , Estabilidade Proteica , Streptococcus pyogenes/metabolismo , Streptococcus pyogenes/patogenicidadeRESUMO
Single-molecule atomic force microscopy and magnetic tweezers experiments have demonstrated that titin immunoglobulin (Ig) domains are capable of folding against a pulling force, generating mechanical work that exceeds that produced by a myosin motor. We hypothesize that upon muscle activation, formation of actomyosin cross bridges reduces the force on titin, causing entropic recoil of the titin polymer and triggering the folding of the titin Ig domains. In the physiological force range of 4-15 pN under which titin operates in muscle, the folding contraction of a single Ig domain can generate 200% of the work of entropic recoil and occurs at forces that exceed the maximum stalling force of single myosin motors. Thus, titin operates like a mechanical battery, storing elastic energy efficiently by unfolding Ig domains and delivering the charge back by folding when the motors are activated during a contraction. We advance the hypothesis that titin folding and myosin activation act as inextricable partners during muscle contraction.
Assuntos
Conectina/metabolismo , Contração Muscular/fisiologia , Músculo Esquelético/metabolismo , Animais , Humanos , Dobramento de ProteínaRESUMO
Proteins fold under mechanical forces in a number of biological processes, ranging from muscle contraction to co-translational folding. As force hinders the folding transition, chaperones must play a role in this scenario, although their influence on protein folding under force has not been directly monitored yet. Here, we introduce single-molecule magnetic tweezers to study the folding dynamics of protein L in presence of the prototypical molecular chaperone trigger factor over the range of physiological forces (4-10 pN). Our results show that trigger factor increases prominently the probability of folding against force and accelerates the refolding kinetics. Moreover, we find that trigger factor catalyzes the folding reaction in a force-dependent manner; as the force increases, higher concentrations of trigger factor are needed to rescue folding. We propose that chaperones such as trigger factor can work as foldases under force, a mechanism which could be of relevance for several physiological processes.Proteins fold under mechanical force during co-translational folding at the ribosome. Here, the authors use single molecule magnetic tweezers to study the influence of chaperones on protein folding and show that the ribosomal chaperone trigger factor acts as a mechanical foldase by promoting protein folding under force.
Assuntos
Proteínas de Bactérias/metabolismo , Proteínas de Escherichia coli/química , Proteínas de Escherichia coli/metabolismo , Peptidilprolil Isomerase/química , Peptidilprolil Isomerase/metabolismo , Dobramento de Proteína , Proteínas de Bactérias/química , Chaperonas Moleculares/química , Chaperonas Moleculares/metabolismo , Análise Espectral/métodosRESUMO
Protein aging may manifest as a mechanical disease that compromises tissue elasticity. As proved recently, while proteins respond to changes in force with an instantaneous elastic recoil followed by a folding contraction, aged proteins break bad, becoming unstructured polymers. Here, we explain this phenomenon in the context of a free energy model, predicting the changes in the folding landscape of proteins upon oxidative aging. Our findings validate that protein folding under force is constituted by two separable components, polymer properties and hydrophobic collapse, and demonstrate that the latter becomes irreversibly blocked by oxidative damage. We run Brownian dynamics simulations on the landscape of protein L octamer, reproducing all experimental observables, for a naive and damaged polyprotein. This work provides a unique tool to understand the evolving free energy landscape of elastic proteins upon physiological changes, opening new perspectives to predict age-related diseases in tissues.
RESUMO
Two variants of the two-component Lantibiotic Lichenicidin, produced by the strains B. Licheniformis VK21 and I89 (Lchα/ Lchß and Bliα/ Bliß peptides, respectively) have been investigated by means of 2 µs-long all-atom molecular dynamics simulations combined with Markov State Models. This rigorous statistical analysis enabled to evaluate the dynamic and kinetic properties of the aforementioned systems which are not accessible via experimental techniques. The structural flexibility characteristic of these small peptides is understood by a delicate equilibrium between random coil, α-helices and ß-sheet structures. The undergoing secondary structure transitions from an α-helix to a ß-sheet observed for Lchα and Lchß peptides, were not present in the Bliα component and provide new insights to understand their mechanism of action.