Reconstruction of low-resolution molecular structures from simulated atomic force microscopy images

BackgroundAtomic Force Microscopy (AFM) is an experimental technique to study structure-function relationship of biomolecules. AFM provides images of biomolecules at nanometer resolution. High-speed AFM experiments produce a series of images following dynamics of biomolecules. To further understand biomolecular functions, information on three-dimensional (3D) structures is beneficial.MethodWe aim to recover 3D information from an AFM image by computational modeling. The AFM image includes only low-resolution representation of a molecule; therefore we represent the structures by a coarse grained model (Gaussian mixture model). Using Monte-Carlo sampling, candidate models are generated to increase similarity between AFM images simulated from the models and target AFM image.ResultsThe algorithm was tested on two proteins to model their conformational transitions. Using a simulated AFM image as reference, the algorithm can produce a low-resolution 3D model of the target molecule. Effect of molecular orientations captured in AFM images on the 3D modeling performance was also examined and it is shown that similar accuracy can be obtained for many orientations.ConclusionsThe proposed algorithm can generate 3D low-resolution protein models, from which conformational transitions observed in AFM images can be interpreted in more detail.General significanceHigh-speed AFM experiments allow us to directly observe biomolecules in action, which provides insights on biomolecular function through dynamics. However, as only partial structural information can be obtained from AFM data, this new AFM based hybrid modeling method would be useful to retrieve 3D information of the entire biomolecule.     

用原子力显微技术研究受体-配体间的相互作用

原子力显微镜(AFM)不仅能对纳米生物结构进行实时动态的形态和结构观察,而且还能以10^-12N(pN)的精度对溶液中生物分子表面的相互作用力进行直接测量,逐渐成为一种研究受体-配体间相互作用的良好工具。本简要综述用AFM研究受体-配体间作用力、受体-配体间相互作用的影响因素及对这些因素的处理方法。     

Atomic force microscopy: a powerful tool to observe biomolecules at work

Atomic force microscopes (AFMs) move a sharp tip attached to a soft cantilever in a TV-raster-like pattern over a surface and record deflections of the tip that correspond to the surface topography. When operated in physiological solutions, an AFM allows biomolecules to be observed in their native environment. Progress in instrumentation, sample-preparation methods and recording conditions has provided images of biomolecules and their assemblies that reveal submolecular details. In addition, the AFM allows conformational changes to be observed directly. This article discusses these points and illustrates them with some pertinent examples.     

原子力显微镜对纳米生物结构的观察和操纵

原子力显微镜不仅能对纳米生物结构进行观察,而且也能对其进行操纵。对纳米生物结构的观察已深入到生物大分子结构水平。原子力显微镜对生物大分子的操纵包括从染色质中提取DNA用于基因分析、对膜蛋白的结构进行观察、对蛋白构象进行可控操纵等。这些纳米技术的应用将揭示生物系统更多的结构和功能信息。     

Single-molecule recognition force spectroscopy of transmembrane transporters on living cells

Atomic force microscopy (AFM) has proven to be a powerful tool in biological sciences. Its particular advantage over other high-resolution methods commonly used is that biomolecules can be investigated not only under physiological conditions but also while they perform their biological functions. Single-molecule force spectroscopy with AFM tip-modification techniques can provide insight into intermolecular forces between individual ligand-receptor pairs of biological systems. Here we present protocols for force spectroscopy of living cells, including cell sample preparation, tip chemistry, step-by-step AFM imaging, force spectroscopy and data analysis. We also delineate critical steps and describe limitations that we have experienced. The entire protocol can be completed in 12 h. The model studies discussed here demonstrate the power of AFM for studying transmembrane transporters at the single-molecule level.     

Probing membrane proteins using atomic force microscopy

To gain insights into how biological molecules function, advanced technologies enabling imaging, sensing, and actuating single molecules are required. The atomic force microscope (AFM) would be one of novel potential tools for these tasks. In this study, techniques and efforts using AFM to probe biomolecules are introduced and reviewed. The state-of-art techniques for characterizing specific single receptor using the functionalized AFM tip are discussed. An example of studying the angiotensin II type 1 (AT1) receptors expressed in sensory neuronal cells by AFM with a functionalized tip is given. Perspectives for identifying and characterizing specific individual membrane proteins using AFM in living cells are provided. Given that many diseases have their roots at the molecular scale and are best understood as a malfunctioning biological nanomachines, the prospects of these unique techniques in basic biomedical research or in clinical practice are beyond our imagination.     

Observing single biomolecules at work with the atomic force microscope

Progress in the application of the atomic force microscope (AFM) to imaging and manipulating biomolecules is the result of improved instrumentation, sample preparation methods and image acquisition conditions. Biological membranes can be imaged in their native state at a lateral resolution of 0.5-1 nm and a vertical resolution of 0. 1-0.2 nm. Conformational changes that are related to functions can be resolved to a similar resolution, complementing atomic structure data acquired by other methods. The unique capability of the AFM to directly observe single proteins in their native environments provides insights into the interactions of proteins that form functional assemblies. In addition, single molecule force spectroscopy combined with single molecule imaging provides unprecedented possibilities for analyzing intramolecular and intermolecular forces. This review discusses recent examples that illustrate the power of AFM.     

Structural and functional imaging with carbon nanotube AFM probes

Atomic force microscopy (AFM) has great potential as a tool for structural biology, a field in which there is increasing demand to characterize larger and more complex biomolecular systems. However, the poorly characterized silicon and silicon nitride probe tips currently employed in AFM limit its biological applications. Carbon nanotubes represent ideal AFM tip materials due to their small diameter, high aspect ratio, large Young's modulus, mechanical robustness, well-defined structure, and unique chemical properties. Nanotube probes were first fabricated by manual assembly, but more recent methods based on chemical vapor deposition provide higher resolution probes and are geared towards mass production, including recent developments that enable quantitative preparation of individual single-walled carbon nanotube tips [J. Phys. Chem. B 105 (2001) 743]. The high-resolution imaging capabilities of these nanotube AFM probes have been demonstrated on gold nanoparticles and well-characterized biomolecules such as IgG and GroES. Using the nanotube probes, new biological structures have been investigated in the areas of amyloid-beta protein aggregation and chromatin remodeling, and new biotechnologies have been developed such as AFM-based haplotyping. In addition to measuring topography, chemically functionalized AFM probes can measure the spatial arrangement of chemical functional groups in a sample. However, standard silicon and silicon nitride tips, once functionalized, do not yield sufficient resolution to allow combined structural and functional imaging of biomolecules. The unique end-group chemistry of carbon nanotubes, which can be arbitrarily modified by established chemical methods, has been exploited for chemical force microscopy, allowing single-molecule measurements with well-defined functionalized tips.     

Atomic force microscopy-based cancer diagnosis by detecting cancer-specific biomolecules and cells

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.     

A general and efficient cantilever functionalization technique for AFM molecular recognition studies

Atomic force microscopy (AFM) is a versatile technique for the investigation of noncovalent molecular associations between ligand–substrate pairs. Surface modification of silicon nitride AFM cantilevers is most commonly achieved using organic trialkoxysilanes. However, susceptibility of the Si? O bond to hydrolysis and formation of polymeric aggregates diminishes attractiveness of this method for AFM studies. Attachment techniques that facilitate immobilization of a wide variety of organic and biological molecules via the stable Si? C bond on silicon nitride cantilevers would be of great value to the field of molecular recognition force spectroscopy. Here, we report (1) the formation of stable, highly oriented monolayers on the tip of silicon nitride cantilevers and (2) demonstrate their utility in the investigation of noncovalent protein–ligand interactions using molecular recognition force spectroscopy. The monolayers are formed through hydrosilylation of hydrogen‐terminated silicon nitride AFM probes using a protected α‐amino‐ω‐alkene. This approach facilitates the subsequent conjugation of biomolecules. The resulting biomolecules are bound to the tip by a strong Si? C bond, completely uniform with regard to both epitope density and substrate orientation, and highly suitable for force microscopy studies. We show that this attachment technique can be used to measure the unbinding profiles of tip‐immobilized lactose and surface‐immobilized galectin‐3. Overall, the proposed technique is general, operationally simple, and can be expanded to anchor a wide variety of epitopes to a silicon nitride cantilever using a stable Si? C bond. © 2012 Wiley Periodicals, Inc. Biopolymers 97: 761–765, 2012.     

Imaging biomolecule arrays by atomic force microscopy.

We describe here a method for constructing ordered molecular arrays and for detecting binding of biomolecules to these arrays using atomic force microscopy (AFM). These arrays simplify the discrimination of surface-bound biomolecules through the spatial control of ligand presentation. First, photolithography is used to spatially direct the synthesis of a matrix of biological ligands. A high-affinity binding partner is then applied to the matrix, which binds at locations defined by the ligand array. AFM is then used to detect the presence and organization of the high-affinity binding partner. Streptavidin-biotin arrays of 100 x 100 microns and 8 x 8 microns elements were fabricated by this method. Contact and noncontact AFM images reveal a dense lawn of streptavidin specific to the regions of biotin derivatization. These protein regions are characterized by a height profile of approximately 40 A over the base substrate with a 350-nm edge corresponding to the diffraction zone of the photolithography. High resolution scans reveal a granular topography dominated by 300 A diameter features. The ligand-bound protein can then be etched from the substrate using the AFM tip, leaving an 8 A shelf that probably corresponds to the underlying biotin layer.     

Atomic force microscopy: from cell imaging to molecular manipulation

The atomic force microscope (AFM) allows to explore the surface of biological samples bathed in physiological solutions, with vertical and horizontal resolutions ranging from nanometers to angstr?ms. Complex biological structures as well as single molecules can be observed and recent examples of the possibilities offered by the AFM in the imaging of intact cells, isolated membranes, membrane model systems and single molecules are discussed in this review. Applications where the AFM tip is used as a nanotool to manipulate biomolecules and to determine intra and intermolecular forces from single molecules are also presented.     

Quantitative characterization of biomolecular assemblies and interactions using atomic force microscopy

Atomic force microscopy (AFM) has been applied in many biological investigations in the past 15 years. This review focuses on the application of AFM for quantitatively characterizing the structural and thermodynamic properties of protein-protein and protein-nucleic acid complexes. AFM can be used to determine the stoichiometries and association constants of multiprotein assemblies and to quantify changes in conformations of proteins and protein-nucleic acid complexes. In addition, AFM in solution permits the observation of the dynamic properties of biomolecular complexes and the measurement of intermolecular forces between biomolecules. Recent advances in cryogenic AFM, AFM on two-dimensional crystals, carbon nanotube probes, solution imaging, high-speed AFM, and manipulation capabilities enhance these applications by improving AFM resolution and the dynamic and operative capabilities of the AFM. These developments make AFM a powerful tool for investigating the biomolecular assemblies and interactions that govern gene regulation.     

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。     

Biomolecular force measurements and the atomic force microscope

The atomic force microscope (AFM) is a surface-sensitive instrument capable of imaging biological samples at nanometer resolution in all environments including liquids. The sensitivity of the AFM cantilever, to forces in the pico Newton range, has been exploited to measure breakaway forces between biomolecules and to measure folding-unfolding forces within single proteins. By attaching specific antibodies to cantilevers the simultaneous imaging of target antigens and identification of antigen-antibody interactions have been demonstrated.     

Detection and localization of single molecular recognition events using atomic force microscopy

Because of its piconewton force sensitivity and nanometer positional accuracy, the atomic force microscope (AFM) has emerged as a powerful tool for exploring the forces and the dynamics of the interaction between individual ligands and receptors, either on isolated molecules or on cellular surfaces. These studies require attaching specific biomolecules or cells on AFM tips and on solid supports and measuring the unbinding forces between the modified surfaces using AFM force spectroscopy. In this review, we describe the current methodology for molecular recognition studies using the AFM, with an emphasis on strategies available for preparing AFM tips and samples, and on procedures for detecting and localizing single molecular recognition events.     

An overview of the biophysical applications of atomic force microscopy

The potentialities of the atomic force microscopy (AFM) make it a tool of undeniable value for the study of biologically relevant samples. AFM is progressively becoming a usual benchtop technique. In average, more than one paper is published every day on AFM biological applications. This figure overcomes materials science applications, showing that 17 years after its invention, AFM has completely crossed the limits of its traditional areas of application. Its potential to image the structure of biomolecules or bio-surfaces with molecular or even sub-molecular resolution, study samples under physiological conditions (which allows to follow in situ the real time dynamics of some biological events), measure local chemical, physical and mechanical properties of a sample and manipulate single molecules should be emphasized.     

Characterization of glucose oxidase immobilization onto mica carrier by atomic force microscopy and kinetic studies

Glucose oxidase (E.C 1.1.3.4) immobilized onto activated surface of mica was analyzed by enzymatic kinetics and visualization with atomic force microscopy (AFM). The activity of the immobilized enzyme decreased with the decrease of concentration of gamma-aminopropyltrimethoxysilane used for the first step of activation of mica, while AFM analysis showed similar homogeneous filling of the surface with the enzyme. The comparison of enzyme activity with its surface filling revealed that there has to be additional vertical structures, which cannot be visualized by the methods of AFM. The simultaneous decrease of the silanizing agent and the concentration of the enzyme led to molecular resolution for the enzyme on the surface of mica. This allows to propose the described method also for analyzing other surfaces of solid materials with coupled biomolecules.     

Equilibrium Sampling for Biomolecules under Mechanical Tension

In the studies of force-induced conformational transitions of biomolecules, the large timescale difference from experiments presents the challenge of obtaining convergent sampling for molecular dynamics simulations. To circumvent this fundamental problem, an approach combining the replica-exchange method and umbrella sampling (REM-US) was developed to simulate mechanical stretching of biomolecules under equilibrium conditions. Equilibrium properties of conformational transitions can be obtained directly from simulations without further assumptions. To test the performance, we carried out REM-US simulations of atomic force microscope (AFM) stretching and relaxing measurements on the polysaccharide pustulan, a (1→6)-β-D-glucan, which undergoes well-characterized rotameric transitions in the backbone bonds. With significantly enhanced sampling convergence and efficiency, the REM-US approach closely reproduced the equilibrium force-extension curves measured in AFM experiments. Consistent with the reversibility in the AFM measurements, the new approach generated identical force-extension curves in both stretching and relaxing simulations—an outcome not reported in previous studies, proving that equilibrium conditions were achieved in the simulations. REM-US may provide a robust approach to modeling of mechanical stretching on polysaccharides and even nucleic acids.     

XPS and AFM analysis of antifouling PEG interfaces for microfabricated silicon biosensors

In the past two decades, the biological and medical fields have seen great advances in the development of biosensors capable of quantifying biomolecules. Many of these biosensors have micro- and nano-scale features, are fabricated using biochip technology, and use silicon as a base material. The creation of antifouling sensor interfaces is critical to avoid serious consequences that arise due to their contact with biological fluids. To this end, we have created thin PEG interfaces of various grafting densities on silicon using a single-step PEG-silane coupling reaction scheme. Initial PEG concentration (5-50 mM) and coupling time (0.5-24 h) were varied to attain different grafting densities, and different PEG interfaces so created were analyzed using XPS and AFM. Furthermore, all the PEG interfaces were evaluated using XPS and AFM for their antifouling abilities using fibrinogen as the model protein. Results indicated that PEG interfaces created in this investigation are appropriate for biosensors with micro- and nano-scale features, and are efficient in controlling protein fouling.