RESUMEN
Endosomal sorting complex required for transport (ESCRT) proteins assemble on the cytoplasmic leaflet of membranes and remodel them. ESCRT is involved in biological processes where membranes are bent away from the cytosol, constricted, and finally severed, such as in multivesicular body formation (in the endosomal pathway for protein sorting) or abscission during cell division. The ESCRT system is hijacked by enveloped viruses to allow buds of nascent virions to be constricted, severed, and released. ESCRT-III proteins, the most downstream components of the ESCRT system, are monomeric and cytosolic in their autoinhibited conformation. They share a common architecture, a four-helix bundle with a fifth helix that interacts with this bundle to prevent polymerizing. Upon binding to negatively charged membranes, the ESCRT-III components adopt an activated state that allows them to polymerize into filaments and spirals and to interact with the AAA-ATPase Vps4 for polymer remodeling. ESCRT-III has been studied with electron microscopy and fluorescence microscopy; these methods provided invaluable information about ESCRT assembly structures or their dynamics, respectively, but neither approach provides detailed insights into both aspects simultaneously. High-speed atomic force microscopy (HS-AFM) has overcome this shortcoming, providing movies at high spatiotemporal resolution of biomolecular processes, significantly increasing our understanding of ESCRT-III structure and dynamics. Here, we review the contributions of HS-AFM in the analysis of ESCRT-III, focusing on recent developments of nonplanar and deformable HS-AFM supports. We divide the HS-AFM observations into four sequential steps in the ESCRT-III lifecycle: (1) polymerization, (2) morphology, (3) dynamics, and (4) depolymerization.
Asunto(s)
Complejos de Clasificación Endosomal Requeridos para el Transporte , Proteínas de la Membrana , Complejos de Clasificación Endosomal Requeridos para el Transporte/metabolismo , Proteínas de la Membrana/metabolismo , Membrana Celular/metabolismo , Microscopía de Fuerza Atómica , Endosomas/metabolismoRESUMEN
Endosomal Sorting Complex Required for Transport III (ESCRT-III) is a conserved protein system involved in many cellular processes resulting in membrane deformation and scission, topologically away from the cytoplasm. However, little is known about the transition of the planar membrane-associated protein assembly into a 3D structure. High-speed atomic force microscopy (HS-AFM) provided insights into assembly, structural dynamics and turnover of Snf7, the major ESCRT-III component, on planar supported lipid bilayers. Here, we develop HS-AFM experiments that remove the constraints of membrane planarity, crowdedness, and support rigidity. On non-planar membranes, Snf7 monomers are curvature insensitive, but Snf7-spirals selectively adapt their conformation to membrane geometry. In a non-crowded system, Snf7-spirals reach a critical radius, and remodel to minimize internal stress. On non-rigid supports, Snf7-spirals compact and buckle, deforming the underlying bilayer. These experiments provide direct evidence that Snf7 is sufficient to mediate topological transitions, in agreement with the loaded spiral spring model.
Asunto(s)
Complejos de Clasificación Endosomal Requeridos para el Transporte , Membrana Dobles de Lípidos , Complejos de Clasificación Endosomal Requeridos para el Transporte/metabolismo , Membrana Dobles de Lípidos/química , Membranas/metabolismo , Microscopía de Fuerza AtómicaRESUMEN
Bacteriorhodopsin is a seven-helix light-driven proton-pump that was structurally and functionally extensively studied. Despite a wealth of data, the single molecule kinetics of the reaction cycle remain unknown. Here, we use high-speed atomic force microscopy methods to characterize the single molecule kinetics of wild-type bR exposed to continuous light and short pulses. Monitoring bR conformational changes with millisecond temporal resolution, we determine that the cytoplasmic gate opens 2.9 ms after photon absorption, and stays open for proton capture for 13.2 ms. Surprisingly, a previously active protomer cannot be reactivated for another 37.6 ms, even under excess continuous light, giving a single molecule reaction cycle of ~20 s-1. The reaction cycle slows at low light where the closed state is prolonged, and at basic or acidic pH where the open state is extended.
Asunto(s)
Bacteriorodopsinas/química , Microscopía de Fuerza Atómica/métodos , Imagen Individual de Molécula/métodos , Biofisica , Citoplasma/metabolismo , Concentración de Iones de Hidrógeno , Transporte Iónico , Cinética , Luz , Simulación de Dinámica Molecular , Nanotecnología , Conformación Proteica , Bombas de Protones/química , Receptores OpioidesRESUMEN
Fast, high-resolution mapping of heterogeneous interfaces with a wide elastic modulus range is a major goal of atomic force microscopy (AFM). This goal becomes more challenging when the nanomechanical mapping involves biomolecules in their native environment. Over the years, several AFM-based methods have been developed to address this goal. However, none of these methods combine sub-nanometer spatial resolution, quantitative accuracy, fast data acquisition speed, wide elastic modulus range and operation in physiological solutions. Here, we present detailed procedures for generating high-resolution maps of the elastic properties of biomolecules and polymers using bimodal AFM. This requires the simultaneous excitation of the first two eigenmodes of the cantilever. An amplitude modulation (AM) feedback acting on the first mode controls the tip-sample distance, and a frequency modulation (FM) feedback acts on the second mode. The method is fast because the elastic modulus, deformation and topography images are obtained simultaneously. The method is efficient because only a single data point per pixel is needed to generate the aforementioned images. The main stages of the bimodal imaging are sample preparation, calibration of the instrument, tuning of the microscope and generation of the nanomechanical maps. In addition, with knowledge of the deformation, bimodal AFM enables reconstruction of the true topography of the surface. It takes ~9 h to complete the whole procedure.
Asunto(s)
Diagnóstico por Imagen de Elasticidad/métodos , Elasticidad , Microscopía de Fuerza Atómica/métodos , Polímeros/química , Proteínas/química , Animales , Materiales Biocompatibles/química , Fenómenos Biomecánicos , Diagnóstico por Imagen de Elasticidad/economía , Diagnóstico por Imagen de Elasticidad/instrumentación , Diseño de Equipo , Halobacterium salinarum/química , Halobacterium salinarum/ultraestructura , Humanos , Microscopía de Fuerza Atómica/economía , Microscopía de Fuerza Atómica/instrumentación , Modelos Moleculares , Complejo de la Endopetidasa Proteasomal/química , Complejo de la Endopetidasa Proteasomal/ultraestructura , Proteínas/ultraestructura , Membrana Púrpura/química , Membrana Púrpura/ultraestructura , Factores de TiempoRESUMEN
Fast quantitative mapping of mechanical properties with nanoscale spatial resolution represents one of the major goals of force microscopy. This goal becomes more challenging when the characterization needs to be accomplished with subnanometer resolution in a native environment that involves liquid solutions. Here we demonstrate that bimodal atomic force microscopy enables the accurate measurement of the elastic modulus of surfaces in liquid with a spatial resolution of 3 Å. The Young's modulus can be determined with a relative error below 5% over a 5 orders of magnitude range (1 MPa to 100 GPa). This range includes a large variety of materials from proteins to metal-organic frameworks. Numerical simulations validate the accuracy of the method. About 30 s is needed for a Young's modulus map with subnanometer spatial resolution.
RESUMEN
The volume of a protein can be estimated from its molecular weight. This approach has also been applied in force microscopy experiments. Two factors contribute to the determination of the volume from a force microscope image, the applied force and the tip radius. Those factors act in opposite directions. Here, we demonstrate that in the optimum conditions to image a protein, the apparent volume deduced from an AFM image overestimates the real protein volume. The lateral broadening due to the tip finite size, makes the simulated volume to exceed the real protein volume value, while the force applied by the tip tends to decrease the measured volume. The measured volume could coincide with the real volume for either a point-size tip at zero force or when the compression exerted by the tip compensates its dilation effects. The interplay between the above factors make unsuitable to apply the molecular weight method to determine the volume of a protein from AFM data.
Asunto(s)
Anticuerpos/química , Humanos , Microscopía de Fuerza Atómica/métodos , Proteínas/químicaRESUMEN
Understanding the mechanical functionalities of complex biological systems requires the measurement of the mechanical compliance of their smallest components. Here, we develop a force microscopy method to quantify the softness of a single antibody pentamer by measuring the stress-strain curve with force and deformation resolutions, respectively, of 5 pN and 50 pm. The curve shows three distinctive regions. For ultrasmall compressive forces (5-75 pN), the protein's central region shows that the strain and stress are proportional (elastic regime). This region has an average Young's modulus of 2.5 MPa. For forces between 80 and 220 pN, the stress is roughly proportional to the strain with a Young's modulus of 9 MPa. Higher forces lead to irreversible deformations (plastic regime). Full elastic recovery could reach deformations amounting to 40% of the protein height. The existence of two different elastic regions is explained in terms of the structure of the antibody central region. The stress-strain curve explains the capability of the antibody to sustain multiple collisions without any loss of biological functionality.
Asunto(s)
Anticuerpos/química , Módulo de Elasticidad , Microscopía de Fuerza Atómica , Estrés MecánicoRESUMEN
A method that combines high spatial resolution, quantitative and non-destructive mapping of surfaces and interfaces is a long standing goal in nanoscale microscopy. The method would facilitate the development of hybrid devices and materials made up of nanostructures of different properties. Here we develop a multifrequency force microscopy method that enables simultaneous mapping of nanomechanical spectra of soft matter surfaces with nanoscale spatial resolution. The properties include the Young's modulus and the viscous or damping coefficients. In addition, it provides the peak force and the indentation. The method does not limit the data acquisition speed nor the spatial resolution of the force microscope. It is non-invasive and minimizes the influence of the tip radius on the measurements. The same tip is used to measure in air heterogeneous interfaces with near four orders of magnitude variations in the elastic modulus, from 1 MPa to 3 GPa.
RESUMEN
The maximum force exerted by the tip of a force microscope on the sample surface is a critical factor that determines the spatial resolution and the degree of invasiveness of the measurement, in particular, on soft materials. Here we determine the conditions needed to image soft matter in the 30-500 MPa range while applying very small forces. Imaging at sub-50 pN in the elastic regime can only be achieved under strict conditions in terms of force constant values (below 0.1 N/m) and free amplitudes (below 2 nm). The peak force depends on the operational parameters, probe properties, the elastic and/or viscoelastic response of the sample, and the contact mechanics model. Images of heterogeneous samples are never taken at a constant peak force. Under the same operational conditions, smaller forces are obtained on the more compliant materials. We also find that the viscoelastic response reduces the peak force with respect to the purely elastic regions. Our findings are summarized in three-dimensional maps that contain the operational conditions for imaging at low forces.