Polymorphism and ultrastructural organization of prion protein amyloid fibrils: an insight from high resolution atomic force microscopy

Amyloid fibrils were produced from the full-length mouse prion protein (PrP) under solvent conditions similar to those used for the generation of synthetic prions from PrP 89-230. Analysis of the ultrastructure by atomic force microscopy revealed extremely broad polymorphism in fibrils formed under a single growth condition. Fibrils varied with respect to the number of constitutive filaments and the manner in which the filaments were assembled. PrP polymerization was found to show several peculiar features: (i) the higher-order fibrils/ribbons were formed through a highly hierarchical mechanism of assembly of lower-order fibrils/ribbons; (ii) the lateral assembly proceeded stepwise; at each step, a semi-stable fibrillar species were generated, which were then able to enter the next level of assembly; (iii) the assembly of lower into higher-order fibrils occurred predominantly in a vertical dimension via stacking of ribbons on top of each other; (iv) alternative modes of lateral association co-existed under a single growth condition; (iv) the fibrillar morphology changed even within individual fibrils, illustrating that alternative modes of filament assembly are inter-convertible and thermodynamically equivalent. The most predominant fibrillar types were classified into five groups according to their height, each of which was divided in up to three subgroups according to their width. Detailed analysis of ultrastructure revealed that the fibrils of the major subtype (height 3.61(+/-0.28)nm, width 31.1(+/-2.0)nm) were composed of two ribbons, each of which was composed of two filaments. The molecular volume calculations indicated that a single PrP molecule occupied a distance of approximately 1.2 nm within a single filament. High polymorphism in fibrils generated in vitro is reminiscent of high morphological diversity of scrapie-associated fibrils isolated from scrapie brains, suggesting that polymorphism is peculiar for polymerization of PrP regardless of whether fibrils are formed in vitro or under pathological conditions in vivo.     

High-resolution atomic force microscopy of soluble Abeta42 oligomers

Soluble oligomers and protofibrils are widely thought to be the toxic forms of the Abeta42 peptide associated with Alzheimer's disease. We have investigated the structure and formation of these assemblies using a new approach in atomic force microscopy (AFM) that yields high-resolution images of hydrated proteins and allows the structure of the smallest molecular weight (MW) oligomers to be observed and characterized. AFM images of monomers, dimers and other low MW oligomers at early incubation times (< 1h) are consistent with a hairpin structure for the monomeric Abeta42 peptide. The low MW oligomers are relatively compact and have significant order. The most constant dimension of these oligomers is their height (approximately 1-3 nm) above the mica surface; their lateral dimensions (width and length) vary between 5 nm and 10nm. Flat nascent protofibrils with lengths of over 40 nm are observed at short incubation times (< or = 3h); their lateral dimensions of 6-8 nm are consistent with a mass-per-length of 9 kDa/nm previously predicted for the elementary fibril subunit. High MW oligomers with lateral dimensions of 15-25 nm and heights ranging from 2-8 nm are common at high concentrations of Abeta. We show that an inhibitor designed to block the sheet-to-sheet packing in Abeta fibrils is able to cap the heights of these oligomers at approximately 4 nm. The observation of fine structure in the high MW oligomers suggests that they are able to nucleate fibril formation. AFM images obtained as a function of incubation time reveal a sequence of assembly from monomers to soluble oligomers and protofibrils.     

原子力显微技术在酶学研究中的应用

酶在生物体的生命活动中占有及其重要的地位,机体功能的和谐统一有赖于酶的作用。原子力显微技术(AFM)作为一门新发展起来的技术,为人们认识酶的结构与功能提供了又一新的窗口。AFM能够在生理条件下对生物样品进行三维成像,在分子水平上实时监测生理生化反应。AFM还能够在皮牛顿精度上测定分子间作用力。目前,AFM已用于单分子酶的化学性质及其作用原理的研究。本简述AFM在酶学中的应用情况。     

原子力显微镜单分子力谱研究生物分子间相互作用

原子力显微镜单分子力谱是近年来发展起来的能在单分子水平研究生物分子相互作用的新工具。本文综述了单分子力谱的测定原理、方法及其在研究蛋白．蛋白、蛋白-DNA相互作用,蛋白质去折叠和活细胞上配体／受体结合中的应用进展。     

High-speed atomic force microscopy: Imaging and force spectroscopy

Atomic force microscopy (AFM) is the type of scanning probe microscopy that is probably best adapted for imaging biological samples in physiological conditions with submolecular lateral and vertical resolution. In addition, AFM is a method of choice to study the mechanical unfolding of proteins or for cellular force spectroscopy. In spite of 28 years of successful use in biological sciences, AFM is far from enjoying the same popularity as electron and fluorescence microscopy. The advent of high-speed atomic force microscopy (HS-AFM), about 10 years ago, has provided unprecedented insights into the dynamics of membrane proteins and molecular machines from the single-molecule to the cellular level. HS-AFM imaging at nanometer-resolution and sub-second frame rate may open novel research fields depicting dynamic events at the single bio-molecule level. As such, HS-AFM is complementary to other structural and cellular biology techniques, and hopefully will gain acceptance from researchers from various fields. In this review we describe some of the most recent reports of dynamic bio-molecular imaging by HS-AFM, as well as the advent of high-speed force spectroscopy (HS-FS) for single protein unfolding.     

Single molecule atomic force microscopy of aerolysin pore complexes reveals unexpected star‐shaped topography

Aerolysin is the paradigmatic member of a large family of toxins that convert from a water‐soluble monomer/dimer into a membrane‐spanning oligomeric pore. While there is x‐ray crystallographic data of its water‐soluble conformation, the most recent structural model of the membrane‐inserted pore is based primarily on data of water‐soluble tetradecamers of mutant protein, together with computational modeling ultimately performed in vacuum. Here we examine this pore model with atomic force microscopy (AFM) of membrane‐associated wild‐type complexes and all‐atom molecular dynamics (MD) simulations in water. In striking contrast to a disc‐shaped cap region predicted by the present model, the AFM images reveal a star‐shaped complex, with a central ring surrounded by seven radial projections. Further, the MD simulations suggest that the locations of the receptor‐binding (D1) domains in the present model are not correct. However, a modified model in which the D1 domains, rather than localized at fixed positions, adopt a wide range of configurations through fluctuations of an intervening linker is compatible with existing data. Thus our work not only demonstrates the importance of directly resolving such complexes in their native environment but also points to a dynamic receptor binding region, which may be critical for toxin assembly on the cell surface. Copyright © 2015 John Wiley & Sons, Ltd.     

Cellular secretion studied by force microscopy

Using the optical microscope, real adventures in cellular research began in earnest in the latter half of the nineteenth century. With the development of the electron microscope, ultramicroscopy, and improved cell staining techniques, significant advances were made in defining intracellular structures at the nanometer level. The invention of force microscopy, the atomic force microscope (AFM) in the mid 1980s, and the photonic force microscope (PFM) in the mid 1990s, finally provided the opportunity to study live cellular structure-function at the nanometer level. Working with the AFM, dynamic cellular and subcellular events at the molecular level were captured in the mid 1990s, and a new cellular structure 'the porosome' in the plasma membrane of all secretory cells has been defined, where specific docking and fusion of secretory vesicles occur. The molecular mechanism of fusion of the secretory vesicle membrane at the base of the porosome membrane in cells, and the regulated release of intravesicular contents through the porosome opening to the extracellular space, has been determined. These seminal discoveries provide for the first time a molecular mechanism of cell secretion, and the possibility to ameliorate secretory defects in disease states.     

Chip-based protein-protein interaction studied by atomic force microscopy

In this article, a technique for accurate direct measurement of protein‐to‐protein interactions before and after the introduction of a drug candidate is developed using atomic force microscopy (AFM). The method is applied to known immunosuppressant drug candidate Echinacea purpurea derived cynarin. T‐cell/CD28 is on‐chip immobilized and B‐cell/CD80 is immobilized on an AFM tip. The difference in unbinding force between these two proteins before and after the introduction of cynarin is measured. The method is described in detail including determination of the loading rates, maximum probability of bindings, and average unbinding forces. At an AFM loading rate of 1.44 × 10⁴ pN/s, binding events were largely reduced from 61 ± 5% to 47 ± 6% after cynarin introduction. Similarly, maximum probability of bindings reduced from 70% to 35% with a blocking effect of about 35% for a fixed contact time of 0.5 s or greater. Furthermore, average unbinding forces were reduced from 61.4 to 38.9 pN with a blocking effect of ～37% as compared with ～9% by SPR. AFM, which can provide accurate quantitative measures, is shown to be a good method for drug screening. The method could be applied to a wider variety of drug candidates with advances in bio‐chip technology and a more comprehensive AFM database of protein‐to‐protein interactions. Biotechnol. Bioeng. 2012; 109: 2460–2467. © 2012 Wiley Periodicals, Inc.     

Ultrastable atomic force microscopy: Improved force and positional stability

Atomic force microscopy (AFM) is an exciting technique for biophysical studies of single molecules, but its usefulness is limited by instrumental drift. We dramatically reduced positional drift by adding two lasers to track and thereby actively stabilize the tip and the surface. These lasers also enabled label-free optical images that were spatially aligned to the tip position. Finally, sub-pN force stability over 100 s was achieved by removing the gold coating from soft cantilevers. These enhancements to AFM instrumentation can immediately benefit research in biophysics and nanoscience.     

Assessing the flexibility of intermediate filaments by atomic force microscopy

Eukaryotic cells contain three cytoskeletal filament systems that exhibit very distinct assembly properties, supramolecular architectures, dynamic behaviour and mechanical properties. Microtubules and microfilaments are relatively stiff polar structures whose assembly is modulated by the state of hydrolysis of the bound nucleotide. In contrast, intermediate filaments (IFs) are more flexible apolar structures assembled from a approximately 45 nm long coiled-coil dimer as the elementary building block. The differences in flexibility that exist among the three filament systems have been described qualitatively by comparing electron micrographs of negatively stained dehydrated filaments and by directly measuring the persistence length of F-actin filaments (approximately 3-10 microm) and microtubules (approximately 1-8 mm) by various physical methods. However, quantitative data on the persistence length of IFs are still missing. Toward this goal, we have carried out atomic force microscopy (AFM) in physiological buffer to characterise the morphology of individual vimentin IFs adsorbed to different solid supports. In addition, we compared these images with those obtained by transmission electron microscopy (TEM) of negatively stained dehydrated filaments. For each support, we could accurately measure the apparent persistence length of the filaments, yielding values ranging between 0.3 microm and 1 microm. Making simple assumptions concerning the adsorption mechanism, we could estimate the persistence length of an IF in a dilute solution to be approximately 1 microm, indicating that the lower measured values reflect constraints induced by the adsorption process of the filaments on the corresponding support. Based on our knowledge of the structural organisation and mechanical properties of IFs, we reason that the lower persistence length of IFs compared to that of F-actin filaments is caused by the presence of flexible linker regions within the coiled-coil dimer and by postulating the occurrence of axial slipping between dimers within IFs.     

Functionalization of amino-modified probes for atomic force microscopy

The probes for atomic force microscopy (AFM) functionalized with bovine serum albumin (BSA) were obtained; they can be used for molecular recognition studies. The procedure of modification and functionalization of the AFM probe included three stages. First, amino probes were obtained by modification in vapors of an amino silane derivative. Then, a covalent bond was formed between the surface amino groups of the probe and a homobifunctional aminoreactive crosslinker. Finally, the probe with a covalently attached crosslinker was functionalized with BSA molecules. The AFM probes were characterized by force measurements at different stages of the modification; the adhesion force and the work of adhesion force were determined. The modification process was confirmed by visualization of BSA and supercoiled pGEMEX DNA molecules immobilized on the standard amino mica and on amino mica modified with a crosslinker.     

Monitoring the process of HypF fibrillization and liposome permeabilization by protofibrils

Much information has appeared in the last few years on the low resolution structure of amyloid fibrils and on their non-fibrillar precursors formed by a number of proteins and peptides associated with amyloid diseases. The fine structure and the dynamics of the process leading misfolded molecules to aggregate into amyloid assemblies are far from being fully understood. Evidence has been provided in the last five years that protein aggregation and aggregate toxicity are rather generic processes, possibly affecting all polypeptide chains under suitable experimental conditions. This evidence extends the number of model proteins one can investigate to assess the molecular bases and general features of protein aggregation and aggregate toxicity. We have used tapping mode atomic force microscopy to investigate the morphological features of the pre-fibrillar aggregates and of the mature fibrils produced by the aggregation of the hydrogenase maturation factor HypF N-terminal domain (HypF-N), a protein not associated to any amyloid disease. We have also studied the aggregate-induced permeabilization of liposomes by fluorescence techniques. Our results show that HypF-N aggregation follows a hierarchical path whereby initial globules assemble into crescents; these generate large rings, which evolve into ribbons, further organizing into differently supercoiled fibrils. The early pre-fibrillar aggregates were shown to be able to permeabilize synthetic phospholipid membranes, thus showing that this disease-unrelated protein displays the same amyloidogenic behaviour found for the aggregates of most pathological proteins and peptides. These data complement previously reported findings, and support the idea that protein aggregation, aggregate structure and toxicity are generic properties of polypeptide chains.     

原子力显微技术成像在生物医学中的应用

原子力显微技术利用探针尖端与标本之间相互作用的力场对标本进行三维成像。这种成像可在生理条件下进行 ,可进行动态观察和标本容易制备是有别于其它成像技术如电子显微镜成像等的特点。对于细胞和生物大分子 ,能够在生理条件下成像具有重要意义。它意味着人们在认识生命本质的方法学方面 ,又向前迈出了新的一步。本文简要综述对细胞和生物大分子的成像在生物医学方面的应用。     

Quasi-linear viscoelastic properties of costal cartilage using atomic force microscopy

Costal cartilage (CC) is one of the load-bearing tissues of the rib cage. Literature on material characterisation of the CC is limited. Atomic force microscopy (AFM) has been extremely successful in characterising the elastic properties of soft biomaterials such as articular cartilage and hydrogels, which are often the material of choice for cartilage models. But AFM data on CC are absent in the literature. In this study, AFM indentations using spherical beaded tips were performed on human CC to isolate the mechanical properties. A novel method was developed for modelling the relaxation indentation experiments based on Fung's quasi-linear viscoelasticity and a continuous relaxation spectrum. This particular model has been popular for uniaxial compression test data analysis. Using the model, the mean Young's modulus of CC was found to be about 2.17, 4.11 and 5.49 MPa for three specimens. A large variation of modulus was observed over the tissue. Also, the modulus values decreased with distance from the costochondral junction.     

Direct visualization of KirBac3.1 potassium channel gating by atomic force microscopy

KirBac3.1 belongs to a family of transmembrane potassium (K⁺) channels that permit the selective flow of K-ions across biological membranes and thereby regulate cell excitability. They are crucial for a wide range of biological processes and mutations in their genes cause multiple human diseases. Opening and closing (gating) of Kir channels may occur spontaneously but is modulated by numerous intracellular ligands that bind to the channel itself. These include lipids (such as PIP₂), G-proteins, nucleotides (such as ATP) and ions (e.g. H⁺, Mg²⁺, Ca²⁺). We have used high-resolution atomic force microscopy (AFM) to examine KirBac3.1 in two different configurations. AFM imaging of the cytoplasmic surface of KirBac3.1 embedded in a lipid bilayer has allowed visualization of the tetrameric assembly of the ligand-binding domain. In the absence of Mg²⁺, the four subunits appeared as four protrusions surrounding a central depression corresponding to the cytoplasmic pore. They did not display 4-fold symmetry, but formed a dimer-of-dimers with 2-fold symmetry. Upon addition of Mg²⁺, a marked rearrangement of the intracellular ligand-binding domains was observed: the four protrusions condensed into a single protrusion per tetramer, and there was an accompanying increase in protrusion height. The central cavity within the four intracellular domains also disappeared on addition of Mg²⁺, indicating constriction of the cytoplasmic pore. These structural changes are likely transduced to the transmembrane helices, which gate the K⁺ channel. This is the first time AFM has been used as an interactive tool to study K⁺ channels. It has enabled us to directly measure the conformational changes in the protein surface produced by ligand binding.     

Metastable and stable states of xanthan polyelectrolyte complexes studied by atomic force microscopy

The compaction of the semiflexible polysaccharide xanthan with selected multi- and polyvalent cations was studied. Polyelectrolyte complexes prepared at concentrations of 1-2 microg/ml were observed by tapping mode atomic force microscopy. High-molecular-weight xanthan compacted with chitosan yields a blend of mainly toroidal and metastable structures and a small fraction of rod-like species. Polyelectrolyte complexes of xanthan with polyethylenimine and trivalent chromium yielded similar structures or alternatively less well packed species. Racquet-type morphologies were identified as kinetically trapped states occurring on the folding path toward the energetically stable state of the toroids. Thermal annealing yielded a shift of the distribution of xanthan-chitosan morphologies toward this stable state. Ensembles of toroidal and rod-like morphologies of the xanthan-chitosan structures, collected using an asphericity index, were analyzed. The mean height of the toroids increased upon heating, with a selective increase in the height range above 2 nm. It is suggested that the observed metastable structures are formed from the high-molecular-weight fraction of xanthan and that these are driven toward the toroidal state, being a low-energy state, following annealing. Considered a model system for condensation of semiflexible polymers, the compaction of xanthan by chitosan captures the system at various stages in the folding toward a low-energy state and thus allows experimental analyses of these intermediates and their evolution.     

Ethambutol-induced alterations in Mycobacterium bovis BCG imaged by atomic force microscopy

Progress in understanding the structure-function relationships of the mycobacterial cell wall has been hampered by its complex architecture as well as by the lack of sensitive, high-resolution probing techniques. For the first time, we used atomic force microscopy (AFM) to image the surface topography of hydrated Mycobacterium bovis bacillus Calmette Guérin cells and to investigate the influence of the antimycobacterial drug ethambutol on the cell wall architecture. While untreated cells showed a very smooth and homogeneous surface morphology, incubation of cells in the presence of ethambutol caused dramatic changes of the fine surface structure. At 4 micro g mL(-1), the drug created concentric striations at the cell surface and disrupted a approximately 8 nm thick cell wall layer, attributed to the outer electron-opaque layer usually seen by electron microscopy, while at 10 micro g mL(-1) an underlying approximately 12 nm thick layer reflecting the thick electron-transparent layer was also altered. These noninvasive ultrastructural investigations provide novel information on the macromolecular architecture of the mycobacterial envelope as well as into the destructuring effects of ethambutol.     

Tapping mode atomic force microscopy of scleroglucan networks

Tapping mode Atomic Force Microscopy (TmAFM) has been used to study the fungal polysaccharide scleroglucan deposited from aqueous solution and dimethyl sulfoxide (DMSO) onto a mica surface. The solutions from which the microscope samples were produced were prepared by first dissolving the solid scleroglucan in 0.1M NaOH, then neutralizing the solution with HCl, followed by dilution to the required concentration in either water or DMSO. It was found that from the aqueous solution described above, scleroglucan forms networks. Based on a comparison of the denatured-renatured and aqueous solution samples, network formation is due to the imperfect registration between the chains forming the triple helices. The relatively large stiffness of the scleroglucan triple helix is also assumed to contribute to the formation of the extended networks. The triple helix diameter was measured to be 0.92 ± 0.27 nm, which is in the same range as that obtained by other researchers using similar techniques. Denatured scleroglucan, deposited from DMSO onto mica, forms a web-like layer on top of which there are sphere-like structures. These morphologies are believed to be due to triple helix denaturation yielding highly flexible single chains in DMSO, which results in coiling and web-like dense packing of scleroglucan upon deposition onto mica. Most interestingly after addition of water to the samples deposited from DMSO, some of the chains can be renatured into short, stiff rod-like structures which are similar to the structures observed by other researchers. The imaging data for aqueous solution deposition can be analyzed by plotting maximum end-to-end distance versus the perimeter of the networks deposited onto mica. This yields a Flory-like exponent of 0.67, which is almost similar in value to that obtained by other researchers for linear structures of scleroglucan but less than that expected for a polymer chain following a self-avoiding walk (v = 0.75) model on a two-dimensional surface. The fractal dimension that can be used to characterize the networks was determined graphically to be 1.22 ± 0.06. © 1997 John Wiley & Sons, Inc. Biopoly 42: 89–100, 1997     

原子力显微镜在细胞弹性研究中的应用

细胞表面的力学性质会随着细胞所处环境的不同而发生改变,它的变化间接反映出胞内复杂的生理过程。原子力显微镜（atomic force microscope,AFM）能以高的灵敏度和分辨率检测活体细胞,通过利用赫兹模型分析力曲线可以获得细胞的弹性信息。本文简介了原子力显微镜的工作原理与工作模式,着重介绍利用AFM力曲线检测细胞弹性的方法及其在细胞运动、细胞骨架、细胞黏附、细胞病理等方面的应用成果,表明AFM已经成为细胞弹性研究中十分重要的显微技术。