This work was supported by the Marie Curie International Reintegration Grant (EP7). P. D. acknowledges the support of the Israel Council for Higher Education.

1.修改提示<ol><li>购买氮化硅（ 的 Si 3 N 4）的AFM悬臂用硅提示（〜2纳米的标称悬臂半径）。 </li><li>通过在无水乙醇中浸泡20分钟，清洁每个AFM悬臂。干燥在常温下。然后通过暴露于O 2等离子体5分钟治疗悬臂。 </li><li>暂停清洁提示上述（3厘米）的含甲基三乙氧基硅烷溶液和3-（氨丙基）三乙氧基硅烷中的15:1的比例（V / V）在惰性气氛（氮气或氩气）下一个干燥器和干燥器连接到真空泵。真空2小时，以形成这两种类型的混合硅烷化合物的的单层。 </li><li>使用金属刀片保持器（用于此工艺制造）放置的尖端的热板上。然后在70℃干燥的大气条件下进行10分钟的提示。在使用前，用乙醇清洗热板，金属支架和镊子。 </li><li>很酷房T提示emperature，然后浸入尖端到在含有0.5％（体积/体积）三乙胺的氯仿5毫浓度芴-PEG-N-羟基琥珀酰亚胺的溶液中（将Fmoc-PEG-NHS，MW 5000道尔顿）1小时室内温度。 </li><li>浸在氯仿中提示5分钟，然后将它浸入二甲基甲酰胺（DMF）额外的5分钟。为了脱保护Fmoc基团所连接的PEG分子，浸在DMF中的20％哌啶（V / V）的尖端30分钟。浸在DMF尖端4分钟，然后在N-甲基-2-吡咯烷酮（NMP）为额外的4分钟。重复顺序浸渍三次。 </li><li>对的氨基酸耦合，浸入尖端到含有N-末端保护的氨基酸（AA）/二异丙基乙胺（DIPEA）/ 2-（1H-苯并三唑-1-基）-1,1,3,3-四甲基脲hexafluophosphate溶液（HBTU），以1:1的摩尔比为1:1，在30毫米的5ml NMP中1.5小时的总浓度。 </li><li>对于肽偶联，浸提示到5 mL溶液续癌宁40毫克被保护的肽的（N末端和侧链，例如将Fmoc-谷氨酰胺（TRT）-Pro - 丙氨酸 - 丝氨酸（TBU）-Ser（TBU）-Arg（PBF）-Tyr（TBU）-COOH）。 ，15毫克2-（1H苯并三唑-1-基）-1,1,3,3-四甲基脲六氟磷酸盐（HBTU），和5ml DIPEA的在NMP 2小时。 </li><li>沾NMP提示4分钟。然后，以由乙酰基保护扩孔空闲/未反应NH 2，以摩尔比4浸在乙酸酐/ DIPEA的混合物30分钟的提示:1和0.65 M在NMP中的总浓度。 </li><li> 用于肽偶联，执行两个额外的步骤。 <ol><li>为了脱保护的肽的侧链，沉浸尖端到含有95％TFA，2.5％三异丙基硅烷和2.5％水1小时的溶液中，然后用氯仿和DMF洗涤。 </li><li>为了除去Fmoc基团的肽的，沉浸尖端到在DMF中的20％哌啶（V / V）30分钟。 </li></ol></li><li>依次蘸肽/氨基酸官能化ð提示各4分钟的DMF（为肽）或NMP（氨基酸），氯仿，50％的乙醇和水。最后干燥在空气中的小费。 </li></ol> 2.表面处理<ol><li>准备云母。用透明胶带切割每次使用前基板（直径9.9毫米）。然后，用三重蒸馏水（TDW）洗涤的表面上。 </li><li>准备二氧化钛涂覆的硅。 <ol><li>切断硅晶片（100）转换成使用金刚石笔2 5cm的正方形。 </li><li>放置在一个15毫升的试管填充有丙酮衬底和超声处理它用于在超声浴五分钟。然后，将这个表面在15毫升的试管填充有异丙醇和超声处理它5分钟。用氮气干燥该衬底。 </li><li>溶解表面活性剂（ 例如 ，BYK-348）的乙醇以制备5％（重量/体积）的溶液中。然后，加入0.02毫升的表面活性剂溶液，以2 30毫升％的TiO 2分散体。 </li><li>从所得到的溶液，滴投0。2毫升干净的Si衬底上。 </li><li>退火在250℃以下滴浇铸表面，在空气中2小时。 41 </li></ol></li></ol> 3.单分子力谱测量<ol><li>用市售的双组分胶附着所需的表面到AFM的金属支架。放置金属支架中的原子力显微镜，其形状为一个小的培养皿的玻璃保持器。填用Tris缓冲液（50mM pH值= 7.4）或任何所希望的培养基此支架。然后，将保持器上的刀片保持器下方的原子力显微镜的阶段。 </li><li>校准用弹簧常数使用具有约10％的绝对不确定度的热波动方法26（包含在AFM软件）从10至30 PN /纳米AFM悬臂。 </li><li>由氨基酸或肽官能尖接近到基片测量的相互作用的力，直到其处于与一压缩基板接触〜200 PN的力，然后立即以各种速度缩回的尖端，从0.1至0.8微米/秒，对于〜200nm的距离。 </li></ol> 4.数据分析<ol><li>乘以光电二极管灵敏度（伏/米），并用实验测定的弹簧常数转换偏转值（V）来强制。 42这是通过AFM软件自动完成。 <ol><li>与一个单分子事件，记录几百曲线（800-1500）获得FD曲线。获得在单分子曲线有两个峰:第一峰值表示尖端与表面和第二个峰对应于与表面分子的特异性相互作用之间的非特异性相互作用。时，将FD曲线含有除这两个峰多，从最可能的力的计算丢弃它们。 注意:仅单个粘附事件都考虑到一个帐户（从10到曲线的30％）。 43 </li></ol></li><lI&gt;为了计算表观负荷率，适合为至少50力与与蜗杆状链（WLC）模型44的距离的曲线只是破裂之前以获得一组负荷率，然后将其用于制备的直方图的明显的负荷率。推导的肽/氨基酸和表面从现行的跳跃之间的解除绑定力从衬底分离所述悬臂之后。 </li></ol>

作者宣称，他们没有竞争的经济利益。

步骤1.3，1.4和1.7中的协议，应广泛护理和在一个非常温和的方式进行。在步骤1.3中，将尖端不应该在用硅烷混合物和硅烷化过程中应在惰性气氛（无水分）进行接触。 45这样做是为了防止多层形成和因硅烷分子容易进行水解在水分存在完成。 45 在步骤1.4中，温度和时间应妥善保管。开始步骤1.5之前，尖端应冷却到室温;否则会被损坏。在偶?...

蛋白质和无机矿物质之间的相互作用导致具有独特性能的复合材料结构。这包括与高的机械强度或独特的光学性质的材料。 1，2例如，与矿物羟磷灰石的胶原蛋白的结合产生任一软或硬的骨骼为不同的功能。 3短肽还可以结合具有高度特异性无机材料。 4，5，6，这些肽的特异性已用于设计新的磁性和电子材料，7，8，9的制造纳米结构材料，生长晶体， 10纳米粒子合成。 11了解底层肽或蛋白质和无机材料之间的相互作用，因此使我们能够具有改进的吸附性能设计新型复合材料的机制。另外，由于植入物具有免疫应答相间是由蛋白质介导，更好地理解与无机材料的蛋白质的相互作用会提高我们的设计的植入物的能力。涉及蛋白质与无机表面相互作用的另一个重要领域是防污材料的制造。 12，13，14，15生物污染是不希望的过程，其中生物附着到表面。这对我们的生活的许多不利影响。例如，在医疗设备的细菌生物污损导致医院获得性感染。海洋生物对船和大型船舶生物污染增加<html>燃料的消耗。 12，16，17，18 单分子力谱（SMFS），使用AFM，可以直接测量的氨基酸或与基板的肽之间的相互作用。 19，20，21，22，23，24，25，26其他方法，如噬菌体展示，27，28 石英晶体微天平（QCM）29或表面等离子体共振（SPR）29，30，31，32，裁判"&gt; 33衡量肽和蛋白质来无机表面的散装的相互作用。34，35，36 这意味着，通过这些方法获得的结果涉及分子或聚集体的合奏。在SMFS，一个或非常少的分子被固定在针尖和它们与所需底物相互作用进行测量。这种方法可以扩展通过从表面拉动蛋白质研究蛋白质折叠。此外，它可以被用来测量细胞和蛋白质与抗体与其配体的结合之间的相互作用。 37，38，39，40本文详细介绍了如何可以肽或氨基酸连接到使用硅醇化学针尖。此外，本文介绍如何执行力测量，以及如何分析结果。

<table><tbody><tr><td>Silicon nitride (Si&lt;sub&gt;3&lt;/sub&gt;N&lt;sub&gt;4&lt;/sub&gt;) AFM cantilevers with silicon tips</td><td>Bruker (Camarilo, CA, USA)</td><td></td><td>MSNL10, nominal cantilevers radius ~2 nm&amp;nbsp;</td></tr><tr><td>Methyltriethoxysilane&amp;nbsp;</td><td>Acros Organics (New Jersey, USA)</td><td></td><td>For Silaylation of the AFM tip&amp;nbsp;</td></tr><tr><td>3-(Aminopropyl) triethoxysilane</td><td>Sigma-Aldrich (Jerusalem, Israel)</td><td></td><td>For Silaylation of the AFM tip</td></tr><tr><td>Triisopropylsilane</td><td>Sigma-Aldrich (Jerusalem, Israel)</td><td></td><td>Used for peptide deprotection</td></tr><tr><td>N-Ethyldiisopropylamine</td><td>Alfa-Aesar (Lancashire, UK)</td><td></td><td>Used for tip modification</td></tr><tr><td>Triethylamine</td><td>Alfa-Aesar (Lancashire, UK)</td><td></td><td>Used for tip modification</td></tr><tr><td>Piperidine</td><td>Alfa-Aesar (Lancashire, UK)</td><td></td><td>Used for tip Fmoc deprotection</td></tr><tr><td>Fluorenylmethyloxycarbonyl-PEG-N-hydroxysuccinimide&amp;nbsp; (Fmoc-PEG-NHS)</td><td>Iris Biotech GmbH (Deutschland, Germany)</td><td></td><td>Used as the covalent flexible linker&amp;nbsp; (MW = 5,000 Da)</td></tr><tr><td>2-(1H-benzotriazol-1-yl)-1,1,3,3,-tetramethyluronium hexafluorophosphate (HBTU)</td><td>Alfa Aser (Heysham, England)</td><td></td><td>Used as a coupling reagent.&amp;nbsp;</td></tr><tr><td>N-methyl-2-pyrrolidone (NMP)</td><td>Acros Organics (New Jersey, USA)</td><td></td><td>Used as Solvent in Tip modification procedure</td></tr><tr><td>DMF (dimethylformamide)</td><td>Merck (Darmstadt, Germany)</td><td></td><td>Used as Solvent in Tip modification procedure</td></tr><tr><td>Chloroform</td><td>Bio-Lab (Jerusalem, Israel)</td><td></td><td>Used as Solvent in Tip modification procedure</td></tr><tr><td>Ethanol (Anhydrous)</td><td>Gadot (Netanya, Israel)</td><td></td><td>Used as Solvent in Tip modification procedure</td></tr><tr><td>Trifluoro acetic acid (TFA)</td><td>Merck (Darmstadt, Germany)</td><td></td><td></td></tr><tr><td>Acetic anhydride</td><td>Merck (Darmstadt, Germany)</td><td></td><td></td></tr><tr><td>Peptides</td><td>GL Biochem (Shanghai, China).</td><td></td><td></td></tr><tr><td>Phenylalanine and Tyrosine&amp;nbsp;</td><td>Biochem (Darmstadt, Germany)&amp;nbsp;</td><td></td><td></td></tr><tr><td>30% TiO&lt;sub&gt;2&lt;/sub&gt; dispersion in the mixture of solvent 2-(2-Methoxyethoxy) ethanol (DEGME) and Ethyl 3-Ethoxypropionate (EEP)</td><td>Applied Vision Laboratories (Jerusalem, Israel)</td><td></td><td>(30%) in the mixture of solvent 2-(2 Methoxyethoxy) ethanol (DEGME) and Ethyl 3-Ethoxypropionate (EEP). Used for TiO&lt;sub&gt;2&lt;/sub&gt; substrate preparation</td></tr><tr><td>Mica substrates</td><td>TED PELLA, INC. (Redding, California, USA)</td><td></td><td>9.9 mm diameter</td></tr></tbody></table>

insights into the interactions of amino acids and peptides with inorganic materials using single-molecule force spectroscopy

蛋白质或肽和无机材料之间的相互作用导致了一些有趣的流程。例如，含矿物质结合蛋白导致具有独特性能的复合材料的形成。此外，生物结垢的不希望的过程是由生物分子，主要是蛋白质，表面上的吸附开始。该有机层是细菌的粘合层，并允许他们与表面相互作用。理解支配在有机 - 无机界面的相互作用的基本力因此对于研究许多领域重要，并可能导致新的材料设计用于光学，机械和生物医学应用。本文展示了单分子力光谱技术，其利用原子力显微镜测量或肽或氨基酸和良好定义的无机表面之间的粘附力。该技术包括一个协议，用于生物分子附着到AFM小费通过原子力显微镜的共价柔性接头和单分子力谱的测量。此外，这些测量值的分析是包括在内。

图1展示尖端修改程序。在第一步骤中，等离子体处理改变氮化硅尖端的表面上。尖端呈现OH基团。然后这些基团将与硅烷反应。在此步骤结束时，尖端的表面将由自由-NH 2基团所覆盖。然后，这些游离胺将用Fmoc -PEG-NHS，共价连接物反应。 Fmoc基团的PEG连接的是由pipyridine，脱保护试剂除去。最后，该检查的氨基酸或肽分子通过使用偶联试剂HBTU PEG的游离...

Watch this Scientific Journal Video about Insights into the Interactions of Amino Acids and Peptides with Inorganic Materials Using Single-Molecule Force Spectroscopy at JoVE.com

Insights into the Interactions of Amino Acids and Peptides with Inorganic Materials Using Single-Molecule Force Spectroscopy

在这里，我们提出了一个协议使用原子力显微镜（AFM）测量由单分子力光谱测量相互作用的定义明确的无机表面和任一的肽或氨基酸之间的力。从测量中获得的信息是重要的，以便更好地了解肽 - 无机材料相间。

洞察氨基酸和多肽与无机材料的使用相互作用的单分子力谱

The interactions between proteins or peptides and inorganic materials lead to several interesting processes. For example, combining proteins with minerals ...

insights-into-interactions-amino-acids-peptides-with-inorganic

Universidad de Cartagena UDEC

Research

JoVE Journal

Chemistry

8.0K 次观看.  The Hebrew University of Jerusalem. 在这里，我们提出了一个协议使用原子力显微镜（AFM）测量由单分子力光谱测量相互作用的定义明确的无机表面和任一的肽或氨基酸之间的力。从测量中获得的信息是重要的，以便更好地了解肽 - 无机材料相间。 

视频: Insights into the Interactions of Amino Acids and Peptides with Inorganic Materials Using Single-Molecule Force Spectroscopy

Multiplexed Single-molecule Force Proteolysis Measurements Using Magnetic Tweezers

The generation and detection of mechanical forces is a ubiquitous aspect of cell physiology, with direct relevance to cancer metastasis1, atherogenesis2 and wound healing3. In each of these examples, cells both exert force on their surroundings and simultaneously enzymatically remodel the extracellular matrix (ECM). The effect of forces on ECM has thus become an area of considerable interest due to its likely biological and medical importance4-7.
	Single molecule techniques such as optical trapping8, atomic force microscopy9, and magnetic tweezers10,11 allow researchers to probe the function of enzymes at a molecular level by exerting forces on individual proteins. Of these techniques, magnetic tweezers (MT) are notable for their low cost and high throughput. MT exert forces in the range of ~1-100 pN and can provide millisecond temporal resolution, qualities that are well matched to the study of enzyme mechanism at the single-molecule level12. Here we report a highly parallelizable MT assay to study the effect of force on the proteolysis of single protein molecules. We present the specific example of the proteolysis of a trimeric collagen peptide by matrix metalloproteinase 1 (MMP-1); however, this assay can be easily adapted to study other substrates and proteases.

In this article we describe the use of magnetic tweezers to study the effect of force on enzymatic proteolysis at the single molecule level in a highly parallelizable manner.

警队复用单分子蛋白水解测量磁性镊子

The  generation and detection of mechanical forces is a ubiquitous aspect of cell physiology,  with direct relevance to cancer metastasis1, atherogenesis2 ...

科研

生物工程

Force Spectroscopy of Single Protein Molecules Using an Atomic Force Microscope

The determination of the folding process of proteins from their amino acid sequence to their native 3D structure is an important problem in biology. Atomic force microscopy (AFM) can address this problem by enabling stretching and relaxation of single protein molecules, which gives direct evidence of specific unfolding and refolding characteristics. AFM-based single-molecule force-spectroscopy (AFM-SMFS) provides a means to consistently measure high-energy conformations in proteins that are not possible in traditional bulk (biochemical) measurements. Although numerous papers were published to show principles of AFM-SMFS, it is not easy to conduct SMFS experiments due to a lack of an exhaustively complete protocol. In this study, we briefly illustrate the principles of AFM and extensively detail the protocols, procedures, and data analysis as a guideline to achieve good results from SMFS experiments. We demonstrate representative SMFS results of single protein mechanical unfolding measurements and we provide troubleshooting strategies for some commonly encountered problems.

We describe the detailed procedures and strategies to measure the mechanical properties and mechanical unfolding pathways of single protein molecules using an atomic force microscope. We also show representative results as a reference for selection and justification of good single protein molecule recordings.

原子力显微镜对单蛋白质分子的力谱

The determination of the folding process of proteins from their amino acid sequence to their native 3D structure is an important problem in biology. ...

生物化学

Investigating Single Molecule Adhesion by Atomic Force Spectroscopy

Atomic force spectroscopy is an ideal tool to study molecules at surfaces and interfaces. An experimental protocol to couple a large variety of single molecules covalently onto an AFM tip is presented. At the same time the AFM tip is passivated to prevent unspecific interactions between the tip and the substrate, which is a prerequisite to study single molecules attached to the AFM tip. Analyses to determine the adhesion force, the adhesion length, and the free energy of these molecules on solid surfaces and bio-interfaces are shortly presented and external references for further reading are provided. Example molecules are the poly(amino acid) polytyrosine, the graft polymer PI-g-PS and the phospholipid POPE (1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoethanolamine). These molecules are desorbed from different surfaces like CH3-SAMs, hydrogen terminated diamond and supported lipid bilayers under various solvent conditions. Finally, the advantages of force spectroscopic single molecule experiments are discussed including means to decide if truly a single molecule has been studied in the experiment.

A protocol to couple a large variety of single molecules covalently onto an AFM tip is presented. Procedures and examples to determine the adhesion force and free energy of these molecules on solid supports and bio-interfaces are provided.

调查单分子粘附的原子力谱

Atomic force spectroscopy is an ideal tool to study molecules at surfaces and interfaces. An experimental protocol to couple a large variety of single ...

工程学

Covalent Immobilization of Proteins for the Single Molecule Force Spectroscopy

In recent years, atomic force microscopy (AFM) based single molecule force spectroscopy (SMFS) extended our understanding of molecular properties and functions. It gave us the opportunity to explore a multiplicity of biophysical mechanisms, e.g., how bacterial adhesins bind to host surface receptors in more detail. Among other factors, the success of SMFS experiments depends on the functional and native immobilization of the biomolecules of interest on solid surfaces and AFM tips. Here, we describe a straightforward protocol for the covalent coupling of proteins to silicon surfaces using silane-PEG-carboxyls and the well-established N-hydroxysuccinimid/1-ethyl-3-(3-dimethyl-aminopropyl)carbodiimid (EDC/NHS) chemistry in order to explore the interaction of pilus-1 adhesin RrgA from the Gram-positive bacterium Streptococcus pneumoniae (S. pneumoniae) with the extracellular matrix protein fibronectin (Fn). Our results show that the surface functionalization leads to a homogenous distribution of Fn on the glass surface and to an appropriate concentration of RrgA on the AFM cantilever tip, apparent by the target value of up to 20% of interaction events during SMFS measurements and revealed that RrgA binds to Fn with a mean force of 52 pN. The protocol can be adjusted to couple via site specific free thiol groups. This results in a predefined protein or molecule orientation and is suitable for other biophysical applications besides the SMFS.

This protocol describes the covalent immobilization of proteins with a heterobifunctional silane coupling agent to silicon-oxide surfaces designed for the atomic force microscopy based single molecule force spectroscopy which is exemplified by the interaction of RrgA (pilus-1 tip adhesin of S. pneumoniae) with fibronectin.

单分子力谱中蛋白质共价固定化的研究

In recent years, atomic force microscopy (AFM) based single molecule force spectroscopy (SMFS) extended our understanding of molecular properties and ...

Investigating Receptor-ligand Systems of the Cellulosome with AFM-based Single-molecule Force Spectroscopy

Cellulosomes are discrete multienzyme complexes used by a subset of anaerobic bacteria and fungi to digest lignocellulosic substrates. Assembly of the enzymes onto the noncatalytic scaffold protein is directed by interactions among a family of related receptor-ligand pairs comprising interacting cohesin and dockerin modules. The extremely strong binding between cohesin and dockerin modules results in dissociation constants in the low picomolar to nanomolar range, which may hamper accurate off-rate measurements with conventional bulk methods. Single-molecule force spectroscopy (SMFS) with the atomic force microscope measures the response of individual biomolecules to force, and in contrast to other single-molecule manipulation methods (i.e.&#160;optical tweezers), is optimal for studying high-affinity receptor-ligand interactions because of its ability to probe the high-force regime (&#62;120 pN). Here we present our complete protocol for studying cellulosomal protein assemblies at the single-molecule level. Using a protein topology derived from the native cellulosome, we worked with enzyme-dockerin and carbohydrate binding module-cohesin (CBM-cohesin) fusion proteins, each with an accessible free thiol group at an engineered cysteine residue. We present our site-specific surface immobilization protocol, along with our measurement and data analysis procedure for obtaining detailed binding parameters for the high-affinity complex. We demonstrate how to quantify single subdomain unfolding forces, complex rupture forces, kinetic off-rates, and potential widths of the binding well. The successful application of these methods in characterizing the cohesin-dockerin interaction responsible for assembly of multidomain cellulolytic complexes is further described.

Cellulosomes are multienzyme complexes designed for digesting cellulose. AFM-based SMFS was used to study the mechanical properties and folding configuration of cellulosome-associated protein assemblies. We present a complete workflow for protein immobilization, data acquisition, and data analysis to study the interactions of individual receptor-ligand complexes involved in cellulosome assembly.

调查纤维素酶与基于AFM的单分子力谱受体 - 配体系统

Cellulosomes are discrete multienzyme complexes used by a subset of anaerobic bacteria and fungi to digest lignocellulosic substrates. Assembly of the ...

Characterizing Individual Protein Aggregates by Infrared Nanospectroscopy and Atomic Force Microscopy

The phenomenon of protein misfolding and aggregation results in the formation of highly heterogeneous protein aggregates, which are associated with neurodegenerative conditions such as Alzheimer&#8217;s and Parkinson&#8217;s diseases. In particular low molecular weight aggregates, amyloid oligomers, have been shown to possess generic cytotoxic properties and are implicated as neurotoxins in many forms of dementia. We illustrate the use of methods based on atomic force microscopy (AFM) to address the challenging task of characterizing the morphological, structural and chemical properties of these aggregates, which are difficult to study using conventional structural methods or bulk biophysical methods because of their heterogeneity and transient nature. Scanning probe microscopy approaches are now capable of investigating the morphology of amyloid aggregates with sub-nanometer resolution. We show here that infrared (IR) nanospectroscopy (AFM-IR), which simultaneously exploits the high resolution of AFM and the chemical recognition power of IR spectroscopy, can go further and enable the characterization of the structural properties of individual protein aggregates, and thus offer insights into the aggregation mechanisms. Since the approach that we describe can be applied also to the investigations of the interactions of protein assemblies with small molecules and antibodies, it can deliver fundamental information to develop new therapeutic compounds to diagnose or treat neurodegenerative disorders.

We describe the application of infrared nanospectroscopy and high-resolution atomic force microscopy to visualize the process of protein self-assembly into oligomeric aggregates and amyloid fibrils, which is closely associated with the onset and development of a wide range of human neurodegenerative disorders.

通过红外纳米光谱和原子力显微镜对单个蛋白质聚合进行特征化

The phenomenon of protein misfolding and aggregation results in the formation of highly heterogeneous protein aggregates, which are associated with ...

OaAEP1-Mediated Enzymatic Synthesis and Immobilization of Polymerized Protein for Single-Molecule Force Spectroscopy

Chemical and bio-conjugation techniques have been developed rapidly in recent years and allow the building of protein polymers. However, a controlled protein polymerization process is always a challenge. Here, we have developed an enzymatic methodology for constructing polymerized protein step by step in a rationally-controlled sequence. In this method, the C-terminus of a protein monomer is NGL for protein conjugation using OaAEP1 (Oldenlandia affinis asparaginyl endopeptidases) 1) while the N-terminus was a cleavable TEV (tobacco etch virus) cleavage site plus an L (ENLYFQ/GL) for temporary N-terminal protecting. Consequently, OaAEP1 was able to add only one protein monomer at a time, and then the TEV protease cleaved the N-terminus between Q and G to expose the NH2-Gly-Leu. Then the unit is ready for next OaAEP1 ligation. The engineered polyprotein is examined by unfolding individual protein domain using atomic force microscopy-based single-molecule force spectroscopy (AFM-SMFS). Therefore, this study provides a useful strategy for polyprotein engineering and immobilization.

Here, we present a protocol to conjugate protein monomer by enzymes forming protein polymer with a controlled sequence and immobilize it on the surface for single-molecule force spectroscopy studies.

OaAEP1介导酶合成和聚合蛋白固定，用于单分子力光谱

Chemical and bio-conjugation techniques have been developed rapidly in recent years and allow the building of protein polymers. However, a controlled ...

Covalent Attachment of Single Molecules for AFM-based Force Spectroscopy

Atomic force microscopy (AFM)-based single molecule force spectroscopy is an ideal tool for investigating the interactions between a single polymer and surfaces. For a true single molecule experiment, covalent attachment of the probe molecule is essential because only then can hundreds of force-extension traces with one and the same single molecule be obtained. Many traces are in turn necessary to prove that a single molecule alone is probed. Additionally, passivation is crucial for preventing unwanted interactions between the single probe molecule and the AFM cantilever tip as well as between the AFM cantilever tip and the underlying surface. The functionalization protocol presented here is reliable and can easily be applied to a variety of polymers. Characteristic single molecule events (i.e., stretches and plateaus) are detected in the force-extension traces. From these events, physical parameters such as stretching force, desorption force and desorption length can be obtained. This is particularly important for the precise investigation of stimuli-responsive systems at the single molecule level. As exemplary systems poly(ethylene glycol) (PEG), poly(N-isopropylacrylamide) (PNiPAM) and polystyrene (PS) are stretched and desorbed from SiOx (for PEG and PNiPAM) and from hydrophobic self-assembled monolayer surfaces (for PS) in aqueous environment.

Covalent attachment of probe molecules to atomic force microscopy (AFM) cantilever tips is an essential technique for the investigation of their physical properties. This allows us to determine the stretching force, desorption force and length of polymers via AFM-based single molecule force spectroscopy with high reproducibility.

基于AFM的力光谱单分子共价附价

Atomic force microscopy (AFM)-based single molecule force spectroscopy is an ideal tool for investigating the interactions between a single polymer and ...

化学

Ensemble Force Spectroscopy by Shear Forces

Single-molecule techniques based on fluorescence and mechanochemical principles provide superior sensitivity in biological sensing. However, due to the lack of high throughput capabilities, the application of these techniques is limited in biophysics. Ensemble force spectroscopy (EFS) has demonstrated high throughput in the investigation of a massive set of molecular structures by converting mechanochemical studies of individual molecules into those of molecular ensembles. In this protocol, the DNA secondary structures (i-motifs) were unfolded in the shear flow between the rotor and stator of a homogenizer tip at shear rates up to 77796/s. The effects of flow rates and molecular sizes on the shear forces experienced by the i-motif were demonstrated. The EFS technique also revealed the binding affinity between DNA i-motifs and ligands. Furthermore, we have demonstrated a click chemistry reaction that can be actuated by shear force (i.e., mechano-click chemistry). These results establish the effectiveness of using shear force to control the conformation of molecular structures.

Ensemble force spectroscopy (EFS) is a robust technique for mechanical unfolding and real-time sensing of an ensemble set of biomolecular structures in biophysical and biosensing fields.

剪切力的集合力谱

Single-molecule techniques based on fluorescence and mechanochemical principles provide superior sensitivity in biological sensing. However, due to the ...

A Fourier Transform Infrared Spectroscopy Technique to Study Peptide Self-Assembly

To prepare a self-assembly solution, dissolve 1 milligram of the lyophilized peptide powder in a mixture of 20% acetonitrile and 10-millimolar HEPES in a 1.5-milliliter microcentrifuge tube. Then, vortex the assembly solution and leave the sample to assemble at room temperature.
When the peptide assembly reaches maturation, dry 8 to 10 microliters of the assembly solution as a thin film on the attenuated total reflection diamond crystal, and monitor the disappearance of a large and broad water peak from 1,640 to 1,630 inverse centimeters as the dry film forms.
Acquire background and infrared spectra from 1,500 to 1,800 inverse centimeters, averaging 50 scans with a 2-inverse centimeter resolution, subtracting the background scans prior to each sample scan.
The infrared signature for beta-sheet assembly should be observed as a sharp peak between 1,625 and 1,635 inverse centimeters.