Sampling Protein Form and Function with the Atomic Force Microscope

To study the structure, function, and interactions of proteins, a plethora of techniques is available. Many techniques sample such parameters in non-physiological environments (e.g. in air, ice, or vacuum). Atomic force microscopy (AFM), however, is a powerful biophysical technique that can probe these parameters under physiological buffer conditions. With the atomic force microscope operating under such conditions, it is possible to obtain images of biological structures without requiring labeling and to follow dynamic processes in real time. Furthermore, by operating in force spectroscopy mode, it can probe intramolecular interactions and binding strengths. In structural biology, it has proven its ability to image proteins and protein conformational changes at submolecular resolution, and in proteomics, it is developing as a tool to map surface proteomes and to study protein function by force spectroscopy methods. The power of AFM to combine studies of protein form and protein function enables bridging various research fields to come to a comprehensive, molecular level picture of biological processes. We review the use of AFM imaging and force spectroscopy techniques and discuss the major advances of these experiments in further understanding form and function of proteins at the nanoscale in physiologically relevant environments.To understand biological processes at the molecular level it is essential to identify the involved proteins and proteinaceous assemblies, to characterize their structure and function, and to unravel their interplay with other proteins and molecules (1). Techniques like x-ray crystallography, electron microscopy, nuclear magnetic resonance spectroscopy, and mass spectrometry have contributed massively to elucidate such protein properties. These techniques can easily sample the properties of a large ensemble of proteins; however, they require subjecting the sample to harsh treatments such as drying, crystallizing, or vaporizing in vacuum, thereby limiting the range of measurable dynamical properties of the sample. One powerful method that permits the investigation of molecules in their native physiological buffer condition is atomic force microscopy (AFM)¹ (2). An atomic force microscope is a microscope and force spectrometer at the same time. The imaging resolution of the atomic force microscope is comparable with that of electron microscopes, and it has the special capability to image samples in a variety of environments such as in vacuum, air, or liquid, which therefore enables studying biological specimens in their native environments (i.e. in buffer solutions) (3, 4). In addition, its ability to “touch” the sample gives it the advantage to manipulate single particles/molecules and probe their mechanical properties (5–8). However, AFM force spectroscopy is currently a technique with rather fast pulling and pushing speeds, thereby often operating out of equilibrium conditions. Improvements with ultrastable atomic force microscopes are underway to tackle this problem with promising results (9, 10). Furthermore, AFM is not well suited to apply and resolve forces at the single piconewton range due to large size tips and relatively stiff cantilevers. The issue of nonspecificity of the tip interaction with the sample is also of concern, especially in pulling experiments that require the capability to accurately recognize and select the appropriate molecule or point of interest. The current introduction of carbon nanotube tips can address the former issue (11, 12), whereas techniques in chemical functionalization can provide directed tip specificity and recognition capability (13–18), thereby further improving and widening the applicability of AFM in the future. In addition, the coupling of the atomic force microscope to fluorescence microscopes further enhances its versatility by adding (single molecule) fluorescence imaging to the AFM imaging capability (19–21), and the development of high speed systems makes it possible for AFM to probe fast dynamics of various biological processes (22–26).The applicability of AFM in proteomics is diverse and includes the characterization of the cell surface proteome (for a recent review, see Ref. 27), label-free detection and counting of single proteins (28, 29), and force spectroscopy measurements of binding and unbinding events (30, 31). In structural biology, AFM has shown to be a powerful tool for high resolution imaging of proteins in near native conditions (3, 6) and structural studies of supramolecular assemblies like protein filaments and viruses by nanoindentation methods (32, 33). These experiments show the potential of AFM to study both “form” and “function” of proteins, thereby resolving questions in proteomics and structural biology quasi-simultaneously. In the following, we will explain the principles of atomic force microscopy and its different operation modes and finally discuss examples of imaging, nanoindentation, and protein (un)binding and unfolding studies using AFM.     

白术多糖WAM-1结构的色谱分析和原子力显微镜观察

通过热水浸提法从草本植物白术根茎提取的水溶性粗多糖,经DEAE-52纤维素柱层析分离和Sephadex G-200凝胶过滤柱层析纯化,得到组分WAM-1.采用高效液相色谱(HPLC)检测WAM-1的纯度,气相色谱(GC)对其单糖组分进行分析,原子力显微镜(AFM)对其分子外貌进行观测.结果显示:WAM-1为均一多糖,由葡萄糖和半乳糖以3.01:1摩尔比构成;在不同浓度溶液条件下,WAM-1分子以不同形态存在,多糖溶液的浓度对WAM-1的分子链构象及链间相互作用形式产生影响,推测可能与WAM-1分子内、分子间的氢键缔合作用有关.多糖浓度为10μg/mL时,可清晰的观察到WAM-1是以刚性链状形态存在,且具有多分支结构.     

Probing the Machinery of Intracellular Trafficking with the Atomic Force Microscope

Atomic force microscopy has emerged as a powerful tool for characterizing single biological macromolecules, macromolecular assemblies, and whole cells in aqueous buffer, in real time, and at molecular-scale spatial and force resolution. Many of the central elements of intracellular transport are tens to hundreds of nanometers in size and highly dynamic. Thus, atomic force microscopy provides a valuable means of addressing questions of structure and mechanism in intracellular transport. We begin this review of recent efforts to apply atomic force microscopy to problems in intracellular transport by discussing the technical principles behind atomic force microscopy. We then turn to three specific areas in which atomic force microscopy has been applied to problems with direct implications for intracellular trafficking: cytoskeletal structure and dynamics, vesicular transport, and receptor–ligand interactions. In each case, we discuss studies which use both intact cellular elements and reconstituted models. While many technical challenges remain, these studies point to several areas where atomic force microscopy can be used to provide valuable insight into intracellular transport at exquisite spatial and energetic resolution.     

原子力显微镜在病毒学研究中的应用

1982年德裔物理学家G.Binnig和H.Rohrer发明了具有原子级分辨率的扫描隧道显微镜(scanning tunneling microscope,STM),使人类第一次能够实时地观察单个原子在物质表面的排列状态和相关的理化性质,两位科学家因此荣获1986年诺贝尔物理学奖[1].在STM基础上发展起来的利用探针扫描技术的一类显微镜统称为扫描探针显微镜(SPM),包括扫描隧道显微镜、原子力显微镜、摩擦力显微镜、磁力显微镜、近场光学显微镜和弹道电子发射显微镜等.档     

原子力显微镜在植物学研究中的应用

原子力显微镜(AFM)是一种探测样品表面信息的有力工具, 它可以在空气和接近样品生理条件下成像, 同时也可以在皮牛(pico-Newton, 10-12 N)至微牛(micro-Newton, 10-6 N)水平上测量力的大小。本文主要介绍了自AFM发明以来, 其在植物大分子、细胞器、细胞、叶片等方面的应用, 并列举了目前 AFM存在的几点不足。     

应用原子力显微镜分析苯甲酸钠生物毒性

应用原子力显微镜(Atomic force microscope, AFM)在单细胞水平上研究食品防腐剂苯甲酸钠(Sodium benzoate, SB)的生物毒性, 从可视化的角度分析了淋巴细胞与不同浓度SB作用不同时间后其形态及其膜超微结构的影响。结果与正常淋巴细胞相比, 随着与淋巴细胞作用的SB浓度和作用时间的增加, 细胞形态及细胞膜明显发生改变, 其超微结构也趋复杂。经SB作用后的细胞高低差Rp-v、均方根粗糙度Rq、平均粗糙度Ra、平均高度4个几何参数值均明显发生改变。对经SB作用后的淋巴细胞进行统计学分析, 并探讨了其作用机制。     

原子力显微镜在生物领域中的应用

原子力显微镜(AFM)作为生物样品表面表征的有力工具,具有独特的优势.本文在介绍原子力显微镜基本原理的基础上,综述了原子力显微镜样品制备以及原子力显微镜形貌分析、力曲线以及动力学分析在生物领域中的应用.     

原子力显微镜在生物领域中的应用

原子力显微镜(AFM)作为生物样品表面表征的有力工具, 具有独特的优势。本文在介绍原子力显微镜基本原理的基础上, 综述了原子力显微镜样品制备以及原子力显微镜形貌分析、力曲线以及动力学分析在生物领域中的应用。     

原子力显微镜在植物学研究中的应用

原子力显微镜（AFM）是一种探测样品表面信息的有力工具,它可以在空气和接近样品生理条件下成像,同时也可以在皮牛（pico-Newton,10^-12N）至微牛（micro—Newton,10^-6N）水平上测量力的大小。本文主要介绍了自AFM发明以来,其在植物大分子、细胞器、细胞、叶片等方面的应用,并列举了目前AFM存在的几点不足。