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. Tip modifica <ol><li> Acquisto nitruro di silicio (Si 3 N 4) cantilever AFM con punte di silicio (raggio sbalzo nominale di ~ 2 nm). </li><li> Pulire ogni cantilever AFM mediante immersione in etanolo anidro per 20 min. Lavaggio a temperatura ambiente. Poi trattare il cantilever esponendoli a O 2 plasma per 5 min. </li><li> Sospendere le punte pulite sopra (3 cm) una soluzione contenente metiltrietossisilano e 3- (amminopropil) triethoxysilane in un rapporto di 15: 1 (v / v) in un essiccatore sotto atmosfera inerte (azoto o argon) e collegare l&#39;essiccatore a una pompa a vuoto. Vuoto per 2 ore per formare un monostrato di questi due tipi di composti silanici misti. </li><li> Usare un supporto punta metallica (fabbricato per questo processo) per posizionare le punte su un piatto caldo. Poi asciugare le punte per 10 minuti a 70 ° C in condizioni atmosferiche. Prima dell&#39;uso, pulire la piastra calda, supporto metallico e pinzette con etanolo. </li><li> Raffreddare le punte a Temperatura, e poi immergere le punte in una soluzione di Fluorenylmethyloxycarbonyl-PEG-N-idrossisuccinimmide (Fmoc-PEG-NHS, MW 5000 Da) ad una concentrazione di 5 mM in cloroformio contenente 0,5% (v / v) trietilammina per 1 ha temperatura ambiente. </li><li> Immergere le punte in cloroformio per 5 minuti e poi immergerlo in dimetilformammide (DMF) per ulteriori 5 min. Per deproteggere il gruppo Fmoc delle molecole di PEG allegate, immergere le punte a 20% piperidina (v / v) in DMF per 30 min. Immergere le punte in DMF per 4 min e poi in N-metil-2-pirrolidone (NMP) per ulteriori 4 min. Ripetere la sequenza immersione per tre volte. </li><li> Per l&#39;accoppiamento di aminoacidi, immergere le punte in una soluzione contenente acido N-terminale protetto amino (AA) / diisopropiletilammina (DIPEA) / 2- (1H-benzotriazolo-1-il) -1,1,3,3-tetramethyluronium hexafluophosphate (HBTU), in un rapporto molare di 1: 1: 1 ad una concentrazione totale di 30 mM in 5 mL NMP per 1,5 h. </li><li> Per l&#39;accoppiamento peptide, immergere le punte in una soluzione cont 5 mLaining 40 mg del peptide protetto (catene terminali e laterali N, ad esempio Fmoc-Gln (Trt) -Pro-Ala-Ser (tBu) -Ser (tBu) -Arg (PBF) -Tyr (tBu) -COOH.) , 15 mg 2- (1H-benzotriazolo-1-il) -1,1,3,3-tetramethyluronium esafluorofosfato (HBTU), e 5 mL di DIPEA in NMP per 2 h. </li><li> Immergere le punte in NMP per 4 min. Poi, per proteggere l&#39;alesatura libera / non reagito NH 2 dal gruppo acetile, immergere le punte per 30 min in una miscela di anidride acetica / DIPEA in un rapporto molare 4: 1 e una concentrazione totale di 0,65 M in NMP. </li><li> Per l&#39;accoppiamento peptide, effettuare due passaggi aggiuntivi. <ol><li> Per deproteggere catene laterali del peptide, immergere le punte in una soluzione contenente 95% TFA 2,5% triisopropylsilane, e acqua 2,5% per 1 h, quindi lavare con cloroformio e DMF. </li><li> Per rimuovere il gruppo Fmoc del peptide, immergere i puntali nel 20% piperidina (v / v) in DMF per 30 min. </li></ol></li><li> Sequenziale immergere il peptide / amino acid-funzionalizzarepunte d per quattro minuti ciascuno in DMF (peptidi) o NMP (per amminoacidi), cloroformio, etanolo al 50% e acqua. Infine asciugare la punta in aria. </li></ol> Preparazione 2. Superficie <ol><li> Preparare mica. Cleave i substrati (diametro 9,9 millimetri) prima di ogni uso utilizzando nastro adesivo. Quindi, lavare le superfici con acqua distillata tripla (TDW). </li><li> Preparare TiO2 silicio Rivestiti. <ol><li> Tagliare il wafer di silicio (100) in 2 cm di lato con una penna di diamante. </li><li> Posizionare il substrato in una provetta da 15 ml di prova riempito con acetone e sonicare per cinque minuti in un bagno ad ultrasuoni. Poi, collocare questa superficie in una provetta da 15 mL di prova riempito con isopropanolo e sonicare per 5 min. Asciugare il substrato con azoto. </li><li> Sciogliere il tensioattivo (per esempio, Byk-348) in etanolo per preparare una soluzione al 5% (peso / volume). Quindi, aggiungere 0,02 ml della soluzione di tensioattivo a 2 mL di 30% TiO 2 dispersione. </li><li> Dalla soluzione risultante, drop-cast 0.2 mL su un substrato di Si pulita. </li><li> Anneal queste superfici goccia pressofuso a 250 ° C per 2 ore in aria. 41 </li></ol></li></ol> 3. singola molecola Forza Spettroscopia Misure <ol><li> Fissare la superficie desiderata ad un supporto metallico del AFM valutata utilizzando colla a due componenti. Posizionare il supporto metallico in supporto di vetro della AFM, che è conformata come una piccola capsula di Petri. Riempire questo supporto con tampone Tris (50 mm pH = 7.4) o qualsiasi mezzo desiderato. Poi, posizionare il supporto sul palco AFM sotto il supporto punta. </li><li> Calibrare i cantilever AFM con costante elastica che variano da 10 a 30 pN / nm utilizzando il metodo termico fluttuazione 26 (incluso nel software AFM) con una incertezza assoluta di circa il 10%. </li><li> Misurare la forza dell&#39;interazione avvicinando l&#39;amminoacido o peptide tip-funzionalizzati al substrato fino a quando è a contatto con il substrato con una compressioneforza di ~ 200 pN e quindi ritrarre subito la punta a varie velocità, da 0,1 a 0,8 micron / s, per una distanza di ~ 200 nm. </li></ol> Analisi 4. I dati <ol><li> Convertire i valori di deflessione (V) per forzare moltiplicando la sensibilità fotodiodo (V / m) e con la costante della molla determinata sperimentalmente. 42 Questo viene fatto automaticamente dal software AFM. <ol><li> Per ottenere curve FD con una singola molecola eventi, registrare diverse centinaia di curve (800-1,500). Ottenere due picchi in una curva singola molecola: il primo picco indica interazioni aspecifiche tra la punta e la superficie e la seconda corrisponde picco alla specifica interazione della molecola con la superficie. Quando la curva FD contiene più di questi due picchi, disfarsene dal calcolo della forza più probabile. NOTA: eventi solo singoli adesione si tenga conto (dal 10 al 30% delle curve). 43 </li></ol></li><li&gt; Per calcolare il tasso di carico apparente, in forma almeno il 50 forza contro curve a distanza con la catena vermiforme (WLC) modello 44 appena prima la rottura di ottenere una serie di tassi di carico, che vengono poi utilizzati per la preparazione di istogrammi i tassi di carico apparenti. Derivare le forze non impegnativa tra le / gli aminoacidi peptidi e la superficie del salto in vigore dopo la separazione del cantilever dal substrato. </li></ol>

Gli autori dichiarano di non avere interessi finanziari concorrenti.

I passi 1.3, 1.4 e 1.7 nel protocollo devono essere effettuate con una vasta cura e in modo molto delicato. Nel passo 1.3, la punta non deve essere in contatto con la miscela silano e il processo di silanizzazione deve essere effettuata in atmosfera inerte (umidità libera). 45 Questo viene fatto al fine di prevenire la formazione di strati e perché le molecole di silano facilmente subiscono idrolisi in presenza di umidità. 45 Nel passo 1.4, ...

L&#39;interazione tra proteine ​​e minerali inorganici porta alla costruzione di materiali compositi con caratteristiche distintive. Questo comprende materiali ad elevata resistenza meccanica e proprietà ottiche uniche. 1, 2, ad esempio, la combinazione del collagene proteina con l&#39;idrossiapatite minerale genera sia ossa molli o duri per diverse funzionalità. 3 peptidi brevi possono anche legare materiali inorganici con elevata specificità. 4, 5, 6 La specificità di questi peptidi sono stati utilizzati per la progettazione di nuovi materiali magnetici ed elettronici, 7, 8, 9 fabbricare materiali nanostrutturati, crescita di cristalli, 10 e sintetizzare le nanoparticelle. 11 Comprendere il meccanismo alla base delle interazioni tra i peptidi o proteine e materiali inorganici sarà quindi permetterà di progettare nuovi materiali compositi con migliorate proprietà di adsorbimento. Inoltre, poiché l&#39;interfase di impianti con una risposta immunitaria è mediata da proteine, una migliore comprensione delle interazioni di proteine ​​con materiali inorganici migliorerà la nostra capacità di progettare impianti. Un&#39;altra area importante che coinvolge le proteine ​​interagenti con superfici inorganiche è la fabbricazione di materiali antivegetative. 12, 13, 14, 15 Biofouling è un processo indesiderabile in cui gli organismi attribuiscono ad una superficie. Esso ha molte implicazioni negative sulle nostre vite. Ad esempio, biofouling di batteri sui dispositivi medici porta a infezioni nosocomiali. Biofouling di organismi marini su barche e grandi navi aumenta la<html> consumo di carburante. 12, 16, 17, 18 Singola molecola di forza spettroscopia (SMFS), utilizzando un AFM, può misurare direttamente le interazioni tra un amminoacido o peptide con un substrato. 19, 20, 21, 22, 23, 24, 25, 26 Altri metodi come phage display, 27, 28 quarzo microbilancia cristallo (QCM) 29 o risonanza plasmonica di superficie (SPR) 29, 30, 31, 32,ref &quot;&gt; 33 misura le interazioni di peptidi e proteine su superfici inorganiche alla rinfusa. 34, 35, 36 Ciò significa che i risultati ottenuti con questi metodi si riferiscono a gruppi di molecole o aggregati. In SMFS, una o poche molecole sono fissati alla punta AFM e le loro interazioni con il substrato desiderato viene misurata. Questo approccio può essere esteso per studiare ripiegamento proteico tirando la proteina dalla superficie. Inoltre, può essere utilizzato per misurare le interazioni tra le cellule e proteine ​​e il legame di anticorpi ai loro ligandi. 37, 38, 39, 40 Questo documento descrive in dettaglio come collegare sia peptidi o amminoacidi fino alla punta AFM con silanolo chimica. Inoltre, il documento spiega come eseguire misurazioni di forza e come analizzare larisultati.

<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

Le interazioni tra proteine ​​o peptidi e materiali inorganici portano a diversi processi interessanti. Per esempio, combinando proteine ​​con minerali porta alla formazione di materiali compositi con proprietà uniche. Inoltre, il processo indesiderabile di biofouling è iniziata dal adsorbimento di biomolecole, principalmente proteine ​​su superfici. Questo strato organico è uno strato di adesione per i batteri e permette loro di interagire con la superficie. La comprensione delle forze fondamentali che governano le interazioni all&#39;interfaccia organico-inorganico è quindi importante per molti settori della ricerca e potrebbe portare alla progettazione di nuovi materiali per applicazioni ottiche, meccaniche e biomediche. Questo documento illustra una tecnica di spettroscopia di forza singola molecola che utilizza un AFM per misurare la forza di adesione tra entrambi peptidi o amminoacidi e superfici inorganiche ben definiti. Questa tecnica comporta un protocollo per fissare la biomolecola alla AFMpunta attraverso un covalente linker flessibile e misure di spettroscopia di forza singola molecola al microscopio a forza atomica. Inoltre, l&#39;analisi di queste misure è incluso.

La figura 1 mostra la procedura di modifica punta. Nella prima fase, un trattamento al plasma cambia la superficie della punta nitruro di silicio. La punta presenta gruppi OH. Questi gruppi poi reagire con i silani. Al termine di questa fase, la superficie della punta sarà coperto da free gruppi -NH 2. Queste ammine libere saranno poi reagire con Fmoc -PEG-NHS, un linker covalente. Il gruppo Fmoc del linker PEG viene rimosso mediante pipyridine, un reagente r...

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

Qui vi presentiamo un protocollo per misurare la forza di interazione fra una superficie inorganica ben definita e sia peptidi o aminoacidi mediante spettroscopia forza singola molecola utilizzando un microscopio a forza atomica (AFM). Le informazioni ottenute dalla misurazione è importante per capire meglio la interfase materiale peptide-inorganico.

Intuizioni le interazioni di aminoacidi e peptidi con materiali inorganici Uso singola molecola Forza Spettroscopia

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 Views.  The Hebrew University of Jerusalem.  Qui vi presentiamo un protocollo per misurare la forza di interazione fra una superficie inorganica ben definita e sia peptidi o aminoacidi mediante spettroscopia forza singola molecola utilizzando un microscopio a forza atomica (AFM). Le informazioni ottenute dalla misurazione è importante per capire meglio la interfase materiale peptide-inorganico. 

Video: 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.

Multiplex singola molecola Forza Misure proteolisi con delle pinzette magnetiche

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

Ricerca

Bioingegneria

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.

Spettroscopia della forza delle singole molecole proteiche utilizzando un microscopio a forza atomica

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

Biochimica

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.

Indagare singola molecola di adesione da Atomic Spectroscopy Forza

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

Ingegneria

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.

Immobilizzazione covalente di proteine per la spettroscopia di forza di singola molecola

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.

Indagare Sistemi recettore-ligando del Cellulosome con sede a AFM singola molecola di Forza Spettroscopia

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.

Caratterizzazione degli aggregati proteici individuali mediante nanospettroscopia a infrarossi e microscopia a forza atomica

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.

Sintesi enzimatica mediata OaAEP1 e immobilizzazione delle proteine polimerizzate per la spettroscopia a singola molecola

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.

Attacco covalente di singole molecole per la spettroscopia della forza basata su AFM

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

Chimica

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.

Spettroscopia Ensemble Force mediante Shear Forces

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.