Name: insights into the interactions of amino acids and peptides with inorganic materials using single-molecule force spectroscopy
Uploaded: 2017-03-06T12:30:00.000+00:00
Duration: 5 min 44 s
Description: 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

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. Sugestão Modificação <ol><li> Compra de nitreto de silício (Si 3 N 4) cantilevers de AFM com pontas de silício (raio cantilever nominal de ~ 2 nm). </li><li> Limpar cada cantilever AFM por imersão em etanol anidro durante 20 min. Seca-se à temperatura ambiente. Em seguida, tratar as consolas por expondo-os a O2 do plasma durante 5 min. </li><li> Suspender as pontas limpas acima (3 cm) contendo uma solução metiltrietoxisilano e 3- (aminopropil) silano em uma proporção de 15: 1 (v / v) em um exsicador sob uma atmosfera inerte (azoto ou árgon) e ligar o exsicador até uma bomba de vácuo. Vácuo durante 2 horas para formar uma monocamada destes dois tipos de compostos de silano misturadas. </li><li> Use um suporte de ponta metálica (fabricada para este processo) para colocar as dicas em uma placa quente. Em seguida, secar as pontas durante 10 minutos a 70 ° C sob condições atmosféricas. Antes do uso, limpe a placa quente, titular metálico e pinças usando etanol. </li><li> Arrefecer as dicas no quarto temperatura, e depois mergulhar as pontas em uma solução de Fluorenilmetiloxicarbonilo-PEG-N-hidroxi-succinimida (Fmoc-PEG-NHS, PM 5000 Da) a uma concentração de 5 mM em clorofórmio contendo 0,5% (v / v) de trietilamina durante 1 h a temperatura do quarto. </li><li> Mergulhe as pontas em clorofórmio durante 5 minutos e, em seguida, mergulhá-lo em dimetilformamida (DMF) durante mais 5 minutos. A fim de desproteger o grupo Fmoc de as moléculas de PEG ligadas, mergulhar as pontas em piperidina a 20% (v / v) em DMF durante 30 min. Mergulhar as pontas em DMF, durante 4 minutos e, em seguida, em N-metil-2-pirrolidona (NMP) para adicional de 4 minutos. Repita o sequencial mergulhar três vezes. </li><li> Para o acoplamento de aminoácidos, imergir as pontas em uma solução contendo N-terminal de amino ácido protegido (AA) / di-isopropiletilamina (DIPEA) / 2- (1H-benzotriazol-1-il) -1,1,3,3-tetrametilurónio hexafluophosphate (HBTU), numa proporção molar de 1: 1: 1 com uma concentração total de 30 mM em 5 mL de NMP durante 1,5 h. </li><li> Para o acoplamento de péptidos, mergulhe as pontas em uma solução cont 5 mLaining 40 mg do péptido protegido (terminal N e cadeias laterais, por exemplo, Fmoc-Gln (Trt) -Pro-Ala-Ser (tBu) -Ser (tBu) -Arg (PBF) -Tyr (tBu) -COOH). , 15 mg de 2- (1H-benzotriazol-1-il) -1,1,3,3-tetrametilurio hexafluorofosfato (HBTU), e 5 ml de DIPEA em NMP durante 2 h. </li><li> Mergulhe as pontas em NMP durante 4 min. Em seguida, para proteger a mandrilagem livre / não reagido NH2 pelo grupo acetilo, mergulhar as pontas durante 30 min numa mistura de anidrido acético / DIPEA a uma razão molar de 4: 1 e uma concentração total de 0,65 M em NMP. </li><li> Para o acoplamento de péptido, realizar duas etapas adicionais. <ol><li> A fim de desproteger as cadeias laterais do péptido, imergir as pontas em uma solução contendo 95% de TFA, 2,5% de tri-isopropilsilano e 2,5% de água durante 1 h, e, em seguida, lava-se com clorofórmio e DMF. </li><li> De modo a remover o grupo Fmoc do péptido, imergir as pontas em piperidina a 20% (v / v) em DMF durante 30 min. </li></ol></li><li> Sequencialmente mergulhar o péptido / amino ácido-funcionalizard dicas para quatro minutos cada em DMF (para péptidos) ou NMP (para os aminoácidos), clorofórmio, 50% de etanol e água. Por fim, seque a ponta no ar. </li></ol> 2. Preparação de Superfície <ol><li> Prepare mica. Clivar os substratos (9,9 mm de diâmetro) antes de cada utilização, usando fita adesiva. Em seguida, lavar as superfícies com água destilada tripla (TPB). </li><li> Prepare TiO 2 -Revestido silício. <ol><li> Cortar o wafer de silício (100) em 2 cm quadrados usando uma caneta de diamante. </li><li> Colocar o substrato num tubo de ensaio de 15 ml cheio com acetona e sonicar durante cinco minutos num banho de ultra-sons. Em seguida, colocar esta superfície de um tubo de ensaio de 15 ml cheio com isopropanol e sonicar durante 5 minutos. Secar o substrato utilizando nitrogênio. </li><li> Dissolve-se o agente tensioactivo (por exemplo, BYK-348) em etanol para preparar uma solução a 5% (peso / volume). Em seguida, adicionar 0,02 ml da solução de agente tensioactivo a uma dispersão de 2 2 ml de 30% de TiO. </li><li> A partir da solução resultante, gota-cast 0.2 mL em um substrato de Si limpa. </li><li> Recozer estas superfícies fundidas-gota a 250 ° C durante 2 h em ar. 41 </li></ol></li></ol> 3. única molécula de medidas de força Espectroscopia <ol><li> Anexar a superfície desejada a um suporte metálico de AFM com cola de dois componentes comercialmente disponíveis. Colocar o suporte da metálico no suporte do copo da AFM, que tem a forma de uma pequena placa de Petri. Preencha este suporte com tampão Tris (pH 50 mM = 7,4) ou qualquer meio desejado. Em seguida, coloque o titular no palco AFM baixo do suporte da ponta. </li><li> Calibrar os braços de suporte de AFM com constantes de mola que variam de 10 a 30 pN / nm, utilizando o método de flutuação térmica 26 (incluído no software AFM) com uma incerteza absoluta de cerca de 10%. </li><li> Medir a força da interacção por se aproximar do aminoácido ou ponta funcionalizada-péptido para o substrato até que esteja em contacto com o substrato com uma compressãoforça de ~ 200 pN e, em seguida, imediatamente retrair a ponta a várias velocidades, de 0,1 a 0,8 ^ m / s, para uma distância de ~ 200 nm. </li></ol> Análise 4. Dados <ol><li> Converter os valores de deformação (V) para forçar multiplicando a sensibilidade fotodíodo (V / m) e utilizando a constante da mola determinado experimentalmente. 42 Isto é feito automaticamente pelo software AFM. <ol><li> Para obter curvas FD com um eventos única molécula, ficha várias centenas de curvas (800-1,500). Obter dois picos numa curva de uma única molécula de: o primeiro pico indica interacções não específicas entre a ponta e a superfície e o segundo corresponde ao pico da interacção específica da molécula com a superfície. Quando, a curva FD conter mais do que estes dois picos, descartá-las a partir do cálculo da força mais provável. NOTA: Apenas eventos de adesão individuais são tidas em conta (de 10 a 30% das curvas). 43 </li></ol></li><li&gt; Para calcular a taxa de carregamento aparente, ajuste de pelo menos 50 força vs. curvas distância com a corrente sem-fim-like (WLC) modelo 44 pouco antes da ruptura para obter um conjunto de taxas de carregamento, que são então utilizados para a preparação de histogramas as taxas de carga aparente. Derivar as forças unbinding entre os péptidos ácidos aminados e / superfície do salto em força depois da separação do raio de acção do substrato. </li></ol>

Os autores declaram que não têm interesses financeiros concorrentes.

Os passos 1.3, 1.4 e 1.7 no protocolo deve ser realizado com cuidado extenso e de uma forma muito suave. No passo 1.3, a ponta não deve estar em contacto com a mistura de silano e o processo de silanização deve ser realizada numa atmosfera inerte (de humidade livre). 45 Isto é feito a fim de evitar a formação de camadas múltiplas e porque as moléculas de silano prontamente sofrer hidrólise na presença de humidade. 45 No passo 1.4, a t...

A interacção entre as proteínas e minerais inorgânicos conduz à construção de materiais compósitos com propriedades distintivas. Isto inclui materiais com elevada resistência mecânica ou as propriedades ópticas únicas. 1, 2, por exemplo, a combinação da proteína de colagénio com a hidroxiapatite mineral, quer gera ossos moles ou duras para diferentes funcionalidades. 3 péptidos curtos, também podem ligar-se materiais inorgânicos com alta especificidade. 4, 5, 6 A especificidade destes péptidos foi usado para a criação de novos materiais magnéticos e electrónicos, 7, 8, 9 fabricar materiais nanoestruturados, crescimento de cristais, 10 e sintetizar nanopartículas. 11 Para entender o mecanismo subjacente interações entre peptídeos ou proteínas e materiais inorgânicos, portanto, nos permitem projetar novos materiais compósitos com melhores propriedades de adsorção. Além disso, uma vez que a interfase de implantes com uma resposta imunitária é mediada por proteínas, uma melhor compreensão das interacções de proteínas com materiais inorgânicos vai melhorar a nossa capacidade para conceber implantes. Outra área importante, que envolve proteínas que interagem com superfícies inorgânicas é a fabricação de materiais anti-incrustações. 12, 13, 14, 15 A incrustação biológica é um processo em que organismos indesejáveis anexar a uma superfície. Ele tem muitas implicações prejudiciais sobre nossas vidas. Por exemplo, biofouling de bactérias sobre dispositivos médicos leva a infecções hospitalares. Biofouling de organismos marinhos em barcos e navios de grande porte aumenta o<html> o consumo de combustível. 12, 16, 17, 18 -Molécula única força espectroscopia (SMFS), usando um AFM, pode medir directamente as interacções entre um aminoácido ou um péptido com um substrato. 19, 20, 21, 22, 23, 24, 25, 26 Outros métodos, como a exibição de fagos, 27, 28 microbalança de cristal de quartzo (QCM) 29 ou ressonância de plasma de superfície (SPR) 29, 30, 31, 32,ref &quot;&gt; 33 medida as interações de peptídeos e proteínas para superfícies inorgânicas em massa. 34, 35, 36 Isto significa que os resultados obtidos por estes métodos referem-se a conjuntos de moléculas ou agregados. Em SMFS, uma ou poucas moléculas são fixadas à ponta de AFM e as suas interacções com o substrato desejado é medido. Esta abordagem pode ser expandido para estudar o dobramento de proteínas, puxando a proteína a partir da superfície. Além disso, ele pode ser usado para medir as interacções entre células e proteínas e a ligação dos anticorpos para os seus ligandos. 37, 38, 39, 40 Este artigo descreve detalhadamente como anexar tanto peptídeos ou aminoácidos para a ponta do AFM usando química silanol. Além disso, o documento explica como realizar medições de força e como analisar aresultados.

<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

As interacções entre as proteínas ou péptidos e materiais inorgânicos levar a vários processos interessantes. Por exemplo, combinando proteínas com minerais conduz à formação de materiais compósitos com propriedades únicas. Além disso, o processo de incrustação biológica indesejável é iniciada pela adsorção de biomoléculas, principalmente proteínas, nas superfícies. Esta camada orgânica é uma camada de adesão de bactérias e que lhes permite interagir com a superfície. Compreender as forças fundamentais que governam as interações na interface orgânica-inorgânica, é importante para muitas áreas de pesquisa e poderia levar à concepção de novos materiais para aplicações ópticas, mecânicas e biomédicas. Este artigo demonstra uma técnica de espectroscopia de força única molécula que utiliza um AFM para medir a força de aderência entre ambos péptidos ou aminoácidos e superfícies inorgânicas bem definidas. Esta técnica envolve um protocolo para a fixação da biomolécula a AFMponta através de um ligante flexível covalente e única molécula de medidas de espectroscopia de força por parte microscópio de força atômica. Além disso, uma análise dessas medidas está incluída.

A Figura 1 apresenta o procedimento de modificação de ponta. No primeiro passo, um tratamento com plasma modifica a superfície da ponta de nitreto de silício. A ponta apresenta grupos OH. Estes grupos serão, em seguida, reagir com os silanos. No final deste passo, a superfície da ponta vai ser coberta por grupos -NH2 livres. Estas aminas livres vai então reagir com Fmoc-PEG-NHS, um ligante covalente. O grupo Fmoc do ligante é removido por PEG pipyridin...

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

Aqui apresentamos um protocolo para medir a força das interações entre uma superfície inorgânica bem definida e tanto peptídeos ou aminoácidos por única molécula de medidas de espectroscopia de força utilizando um microscópio de força atômica (AFM). As informações obtidas a partir da medição é importante para entender melhor a interfase material de peptídeo-inorgânico.

Insights sobre as interações de aminoácidos e peptídeos com Materiais Inorgânicos Usando Single-Molecule Força Spectroscopy

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

Research

JoVE Journal

Chemistry

8.0K visualizações.  The Hebrew University of Jerusalem.  Aqui apresentamos um protocolo para medir a força das interações entre uma superfície inorgânica bem definida e tanto peptídeos ou aminoácidos por única molécula de medidas de espectroscopia de força utilizando um microscópio de força atômica (AFM). As informações obtidas a partir da medição é importante para entender melhor a interfase material de peptídeo-inorgânico. 

Vídeo: 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.

Multiplexados molécula única Força Medidas proteólise, usando uma pinça magnética

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

Bioengenharia

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.

Espectroscopia de força das moléculas de proteína único, usando um microscópio de força atômica

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

Bioquímica

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.

Investigando única molécula de adesão de Força Atômica 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 ...

Engenharia

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.

Imobilização covalente de proteínas para a espectroscopia de força única molécula

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.

Investigando Sistemas receptor-ligante do Celulossoma com base-AFM única molécula Força Espectroscopia

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.

Caracterização de agregados proteicos individuais por Nanoespectroscopia infravermelha e microscopia de força atômica

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.

Síntese Enzimática Mediada oOAEP1 e Imobilização de Proteína Polimerizada para Espectroscopia de Força De Molécula Única

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.

Ligação covalente de moléculas únicas para espectroscopia de força baseada em AFM

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

Química

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.

Espectroscopia de Força de Ensemble por Forças de Cisalhamento

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.