The authors would like to acknowledge the funding received from the American Heart Association (SDG12SDG9130039).

1. Ponteggio liposomi Preparazione NOTA: Idealmente, esperimenti iniziali dovrebbe usare un&#39;impalcatura relativamente semplice e informe (composto da DOPC (1,2-dioleoyl- SN -glycero-3-phosphocholine o PC) e DGS-NTA (Ni 2+) (1,2-dioleoyl - sn -glycero-3 - [(N - (5-ammino-1-carbossipentil) acido imminodiacetico) succinil]. (nickel sale)) Edificio fuori di questi esperimenti, carica lipidi, flessibilità e curvatura può essere introdotto come singoli fattori con il potenziale per alterare le interazioni membrana-prossimale. Queste modifiche possono essere realizzati aggiungendo quantità definite di specifici componenti lipidici, inclusi fosfatidilserina o cardiolipina (CL), fosfatidiletanolammina (DOPE o PE), o galactosyl ceramide (β). <ol><li> Combinare lipidi disciolti in cloroformio in una provetta di vetro pulito. Evaporare il solvente con azoto secco, mentre la rotazione del tubo per formare un sottile film lipidico. Rimuovere solvente residuo con una centrifugal evaporatore per 1 ora a 37 ° C. NOTA: Varie formulazioni di liposomi vengono utilizzati nei protocolli di seguito descritto: liposomi ponteggi (3,3 mol% DGS-NTA (Ni 2+) / 96,7 mol% DOPC), liposomi ponteggi con cardiolipina (3,3 mol% DGS-NTA (Ni 2+) / 10 mol% cardiolipina / 86,7 mol% DOPC), liposomi ponteggio flessibili con cardiolipina (3.3 mol% DGS-NTA (Ni 2+) / 10% mol cardiolipina / 35 mol% DOPE / 51,7 mol% DOPC), e impalcatura arricchito liposomi (10 mol% DGS-NTA (Ni 2+) / 15% mol cardiolipina / 35 mol% DOPE / 40 mol% DOPC). </li><li> Aggiungere tampone A (25 mM HEPES (4- (2-idrossietil) -1-acido piperazineethanesulfonic), 150 mM KCl, pH regolato a 7,5 con KOH) preriscaldato a 37 ° C in modo che la concentrazione di lipidi finale 1 - 2 mM. Incubare 30 minuti a 37 ° C con vortex occasionale per risospendere completamente la miscela lipidica (Figura 1a). </li><li> Trasferire in una provetta di plastica, porre il tubo in azoto liquido fino a completo congelamento (roughly 30 s), e posto in un bagno d&#39;acqua C 37 ° fino a quando completamente scongelati (circa 1 - 2 min). Ripetere per un totale di 4 cicli di gelo-disgelo (Figura 1B). </li><li> Preparare un estrusore lipidico immergendo 4 supporti del filtro e un filtro di policarbonato in tampone e assemblare l&#39;estrusore secondo le istruzioni del produttore. Estrudere la soluzione lipidica attraverso il filtro 21 volte. Utilizzare dolce, pressione costante per garantire una distribuzione di dimensione omogenea (Figura 1c). NOTA: Per tutti gli esperimenti descritti in questo protocollo, un filtro 1,0 micron in policarbonato è stato utilizzato per l&#39;estrusione. Interazione DRP1 con lipidi anionici può essere osservata con una varietà di diametri di liposomi che variano da 50 nm a 400 nm 12 o superiore 13. Quindi, la dimensione del filtro di 1 micron è stato scelto per essere ideale sia GTPasi attività e per la microscopia elettronica. Se altri diametri di liposomi sono desiderati, preparazione dei giganti vescicole unilamellari 14,15 (GUVs) o piccolo unilamellar vescicole 16 (SUV) può essere utilizzato. Diffusione della luce dinamica può essere utilizzato per valutare l&#39;eterogeneità liposomi dimensioni 13. </li><li> Conservare liposomi estrusi a 4 ° C e scartare dopo 3-5 d. </li></ol> 2. L&#39;utilizzo di ponteggi liposomi per la Proteina Analisi Binding <ol><li> Preparazione del campione <ol><li> Incubare His-tag MffΔTM (5 micron finale) con liposomi ponteggi (40 mol% PC / 35 mol% PE / 15 mol% CL / 10 mol% DGS-NTA (Ni 2+); 50 um finale) per almeno 15 min a RT in tampone a + BME (25 mM HEPES, 150 mM KCl, 10 mM β-mercaptoetanolo (BME), pH regolato a 7,5 con KOH). Per un controllo QFP-libera, incubare liposomi con il suo tag-proteina di controllo (come la GFP) per legare e scudo esposto NTA (Ni 2+). NOTA: MffΔTM è stato espresso e purificato come descritto in un precedente studio 2. GFP è stato purificato in modo analogo, ma il passo di scambio ionico è stato omesso. BME è stato richiesto per questi experiments perché DRP1 è sensibile all&#39;ossidazione, che possono modificare le sue proprietà di attività e di assemblaggio. </li><li> Aggiungere DRP1 (2 mM finale) e incubare per 1 ora a temperatura ambiente. NOTA: DRP1 è stato espresso e purificato come descritto in un precedente studio 2. Dopo incubazione con DRP1, l&#39;effetto vincolante per deformazione della membrana nucleotide può essere studiata incubando un&#39;altra ora con 2 mM MgCl 2 e sia 1 mM GTP, 1 mM GMP-PCP, o tampone A + BME. </li></ol></li><li> Negativo Stain microscopia elettronica a trasmissione (EM) Analisi <ol><li> Transfer 5 microlitri di campione ad un foglio di pellicola di laboratorio, e stabilire una griglia Cu / Rh carbonio rivestito sul campione. Incubare la griglia 1 min sul campione, cancellare via il liquido in eccesso su carta da filtro, e trasferire ad un calo del 2% acetato di uranile. Incubare 1 min, tamponare l&#39;eccesso macchia sulla carta da filtro, e trasferire a una casella della griglia. Conservare sotto vuoto O / N per garantire la piena essiccazione. </li><li> campioni di immagine utilizzando un microsc elettronico a trasmissioneope a 18.500 - 30,000X ingrandimento per osservare i cambiamenti ultrastrutturali di proteine e di liposomi morfologie 17. Nota: le modifiche ultrastrutturali possono essere quantificati utilizzando immagine software di analisi, come ad esempio ImageJ 13 (http://imagej.nih.gov/ij/). decorazione di proteine ​​può essere misurata se confrontato con i modelli di lipidi nudi. Inoltre, i diametri dei segmenti tubolari possono essere misurati dalla porzione più esterna dei complessi 13. Un&#39;analisi più dettagliata può essere effettuata utilizzando crio-microscopia elettronica 17. Questo metodo può essere usato per immagini complessi proteina-lipidi nativi in ​​solvente senza l&#39;uso di coloranti metalli pesanti che rivestono il campione. In questo modo, le caratteristiche strutturali dettagliate non apparenti macchia negativo, inclusi i cambiamenti nella morfologia dei lipidi di fondo, possono essere esaminate e quantificati. </li></ol></li></ol> 3. L&#39;uso del ponteggio liposomi per enzimatica Assay Nota: A colorisaggio GTPase metrica 18 è stato utilizzato per misurare la liberazione di fosfato tramite GTP idrolisi. Saggi GTPasi alternativi sono disponibili 19 e possono essere implementati in base alle esigenze. <ol><li> Incubare His-tag MffΔTM (QFP), Fis1ΔTM (Fis1), o GFP (5 micron finale per tutti) con liposomi ponteggio (150 micron finali) per 15 minuti a temperatura ambiente in tampone A + BME (volume = 30 ml). Aggiungere DRP1 (500 nM finale) e incubare un ulteriore 15 minuti a temperatura ambiente (volume = 80 ml). NOTA: Fis1 è stato purificato in modo analogo a MFF 2, ma la fase di cromatografia a scambio ionico è stato omesso. Lo scopo della sua tag-GFP è quello di proteggere il NTA (Ni 2+) headgroups e prevenire le interazioni di carica non specifiche con altre proteine. Se nessun effetto si osserva in assenza di GFP, allora questo controllo può non essere necessaria. Alternativi proteine ​​bloccaggio (di dimensioni paragonabili alla proteina di interesse) possono essere usati pure, ma GFP permette la visualizzazione diretta delle interazioni con scafpiegare liposomi. </li><li> Tubi Trasferire un termociclatore impostato a 37 ° C, e avviare le reazioni di aggiunta di GTP e MgCl 2 (1 mM e 2 mM finale, rispettivamente; volume = 120 ml). </li><li> A intervalli di tempo desiderati (cioè T = 5, 10, 20, 40, 60 min), trasferire 20 microlitri di reazione nei pozzetti della micropiastra contenente 5 ml di 0,5 M EDTA per chelare Mg 2+ e fermare la reazione. </li><li> Preparare una serie di standard di fosfato diluendo KH 2 PO 4 in tampone A + BME per calibrare i risultati. Un utile set di standard è 100, 80, 60, 40, 20, 10, 5 e 0 pM. Aggiungere 20 ml di ciascuno per pozzetti contenenti 5 ml di 0,5 M EDTA. </li><li> Aggiungere 150 ml di malachite verde reagente (1 mm di malachite verde carbinolo, 10 mM ammonio molibdato tetraidrato, e 1 N HCl) in ogni pozzetto, e leggere OD 650 5 min dopo l&#39;aggiunta. NOTA: GTP è acido labile e si idrolizzano in presenza di reagente verde malachite. Assicurarsi che tha il tempo tra l&#39;aggiunta del reagente di malachite e la lettura è costante per garantire la riproducibilità dei risultati. </li><li> Generare una curva standard tracciando OD 650 degli standard in funzione della concentrazione di fosfato. Utilizzare la regressione lineare per determinare la relazione tra OD 650 e la concentrazione di fosfato in un campione. </li><li> Utilizzando la regressione lineare, convertire la OD 650 dei campioni di reazione proteina mM fosfato. Determinare la velocità di generazione di fosfato per ciascuna miscela di reazione riportando concentrazione di fosfato in funzione del tempo, e convertire k cat dividendo il tasso dalla concentrazione DRP1 (0,5 mM). NOTA: Solo la velocità lineare iniziale dovrebbe essere usato per determinare la velocità di generazione di fosfato, e un minimo di 3 punti di dati deve essere utilizzato. Se la velocità di reazione è sufficientemente rapida che i primi tre punti di dati non sono lineari (cioè la r 2 del fit lineare è inferiore a 0,9) di un segnoNaturalmente tempo ificantly breve con almeno 3 punti di tempo deve essere eseguita. </li></ol>

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The authors have nothing to disclose.

Questo protocollo offre un metodo per studiare le interazioni proteina-proteina che coinvolgono proteine ​​integrali di membrana. Utilizzando un ponteggio liposoma modulare, investigatori sono in grado di valutare l&#39;attività di una o più proteine ​​in un ambiente lipidico-prossimale. Studi precedenti hanno dimostrato un metodo simile per gli enzimi del recettore della membrana plasmatica 24 - 26. Abbiamo ampliato questo metodo per incorporare cofattori lipidici ed esplorare...

Studiare le interazioni proteina-proteina di membrana-prossimale è uno sforzo impegnativo a causa della difficoltà di ricapitolare l&#39;ambiente nativo delle proteine integrali di membrana coinvolte 1. Ciò è dovuto alla necessità di detersivo solubilizzazione e l&#39;orientamento incoerente delle proteine ​​in proteoliposomi. Per evitare questi problemi, abbiamo utilizzato una strategia quale i domini solubili di proteine integrali di membrana sono espressi come proteine di fusione His-tag, e questi frammenti solubili sono ancorati liposomi ponteggio tramite interazioni con NTA (Ni 2+) headgroups al lipide superficie. L&#39;utilizzo di questi ponteggi, interazioni proteina-lipidi prossimale possono essere indagati in un intervallo di lipidi e proteine ​​composizioni. Abbiamo effettivamente applicato questo metodo per studiare le interazioni critiche proteina-proteina che regolano il montaggio del complesso fissione mitocondriale ed esaminare le interazioni lipidi che modulano questo process 2. Durante la fissione mitocondriale, una conservata proteina rimodellamento della membrana, chiamata Dynamin-related protein 1 (DRP1) 3, viene reclutato per la superficie del mitocondriale esterna della membrana (OMM) in risposta a segnali cellulari che regolano l&#39;omeostasi energetica, segnale apoptotico, e molti altri processi mitocondriali integrali. Questo grande, GTPase citosolica viene reclutato alla superficie dei mitocondri attraverso le interazioni con proteine OMM integrali 4 - 8. Il ruolo di una tale proteina, mitocondriale fissione Factor (QFP), è stato difficile chiarire causa di una debole interazione evidente con DRP1 in vitro. Tuttavia, studi genetici hanno chiaramente dimostrato che QFP è essenziale per il successo 7,8 fissione mitocondriale. Il metodo descritto in questo manoscritto è stato in grado di superare le carenze precedenti introducendo le interazioni lipidi simultanee che promuovono le interazioni DRP1-QFP. Nel complesso, questo nuovo metodo di analisi REVEAportato interazioni fondamentali che guidano il montaggio del complesso fissione mitocondriale e ha fornito una nuova fase per gli studi strutturali e funzionali in corso di questa macchina molecolare essenziale. Fino ad oggi, l&#39;esame delle interazioni tra DRP1 e QFP sono state complicate dalla flessibilità intrinseca del QFP 9, l&#39;eterogeneità dei polimeri DRP1 2,10, e la difficoltà di purificazione e di ricostituzione full-length QFP con un dominio transmembrana intatto 11. Abbiamo affrontato queste sfide utilizzando liposomi NTA (Ni 2+) ponteggio per ricostituire His-tag QFP manca il suo dominio transmembrana (MffΔTM-His 6). Questa strategia è stata vantaggioso perché MffΔTM era estremamente solubile quando sovra-espresso in E. coli, e questa proteina isolata è stato facilmente ricostituiti in liposomi patibolo. Quando legato a questi modelli di lipidi, MFF ha assunto un identico, rivolto verso l&#39;esterno orientamento sulla superficie della membrana.Oltre a questi vantaggi, lipidi mitocondriali, come cardiolipina, sono stati aggiunti per stabilizzare QFP piegatura e di associazione con la membrana 11. Cardiolipin interagisce anche con il dominio variabile della DRP1 2,12 che può stabilizzare questa regione disordinato e facilitare il montaggio della macchina fissione. Questo metodo robusto è ampiamente applicabile per gli studi futuri che cercano di valutare le interazioni delle proteine ​​di membrana-prossimale. Attraverso l&#39;uso di ulteriori interazioni tethering / affinità, la sofisticazione di questi studi ricostituzione membrana può essere migliorato per imitare ulteriori complessità trovato alla superficie delle membrane all&#39;interno delle cellule. Allo stesso tempo, composizioni lipidiche possono essere modificati per imitare più accuratamente gli ambienti nativi di questi complessi macromolecolari. In sintesi, questo metodo fornisce un mezzo per esaminare i relativi contributi di proteine ​​e lipidi nel plasmare morfologie membrana a durante proc cellulare criticaesses.

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			<td height="21" style="height:21px;width:287px;">Refrigerated CentriVap Concentrator</td>
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			<td height="21" style="height:21px;width:287px;">VersaMax Microplate reader</td>
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using scaffold liposomes to reconstitute lipid-proximal protein-protein interactions in vitro

Studi di proteine integrali di membrana in vitro sono spesso complicata dalla presenza di un dominio transmembrana idrofobico. A complicare ulteriormente questi studi, reintegrazione delle proteine ​​di membrana detergente-solubilizzato in liposomi è un processo stocastico in cui topologia di proteine ​​è impossibile da far rispettare. Questo articolo offre un metodo alternativo per queste tecniche impegnative che utilizza un ponteggio a base di liposomi. Protein solubilità è esaltata dalla delezione del dominio transmembrana, e questi amminoacidi sono sostituiti con un residuo tethering, ad esempio un His-tag. Questo tether interagisce con un gruppo di ancoraggio (Ni 2+ coordinato da acido nitrilotriacetico (NTA (Ni 2+)) per His-tag proteine), che impone una topologia proteine uniforme sulla superficie del liposoma. Un esempio è presentato in cui l&#39;interazione tra proteine ​​Dynamin legati 1 (DRP1) con una proteina integrale di membrana, mitocondriale fissione Factor (QFP), era investigated utilizzando questo metodo impalcatura liposomi. In questo lavoro, abbiamo dimostrato la capacità di QFP di reclutare in modo efficiente DRP1 solubile alla superficie dei liposomi, che ha stimolato la sua attività GTPasi. Inoltre, DRP1 era in grado di tubulate il modello di lipidi MFF-decorato in presenza di lipidi specifici. Questo esempio dimostra l&#39;efficacia di liposomi impalcature utilizzando saggi strutturali e funzionali e mette in evidenza il ruolo del QFP nella regolazione dell&#39;attività DRP1.

Mentre l&#39;interazione tra DRP1 e QFP ha dimostrato di essere importante per fissione mitocondriale, questa interazione è stato difficile ricapitolare in vitro. Il nostro obiettivo era quello di emulare meglio l&#39;ambiente cellulare in cui DRP1 e del QFP interagiscono. A tal fine, liposomi contenenti concentrazioni limitanti del NTA (Ni 2+) headgroups sono stati preparati mediante reidratazione un film lipidico come descritto sopra. La soluzione lipidica inizialm...

Watch this Scientific Journal Video about Using Scaffold Liposomes to Reconstitute Lipid-proximal Protein-protein Interactions In Vitro at JoVE.com

Using Scaffold Liposomes to Reconstitute Lipid-proximal Protein-protein Interactions In Vitro

This paper describes a method for assessing the interactions and assemblies of integral membrane proteins in vitro with various partner factors in a lipid-proximal environment.

Utilizzando impalcatura liposomi per ricostituire interazioni proteina-proteina lipidi-prossimale<em&gt; In Vitro</em

Studies of integral membrane proteins in vitro are frequently complicated by the presence of a hydrophobic transmembrane domain. Further complicating ...

using-scaffold-liposomes-to-reconstitute-lipid-proximal-protein

Research

JoVE Journal

Biochemistry

Using Scaffold Liposomes to Reconstitute Lipid-proximal Protein-protein Interactions In Vitro

8.7K Views.  Case Western Reserve University School of Medicine. This paper describes a method for assessing the interactions and assemblies of integral membrane proteins in vitro with various partner factors in a lipid-proximal environment. 

Video: Using Scaffold Liposomes to Reconstitute Lipid-proximal Protein-protein Interactions In Vitro

Fluorescence Anisotropy as a Tool to Study Protein-protein Interactions

Protein-protein interactions play an essential role in the function of a living organism. Once an interaction has been identified and validated it is necessary to characterize it at the structural and mechanistic level. Several biochemical and biophysical methods exist for such purpose. Among them, fluorescence anisotropy is a powerful technique particularly used when the fluorescence intensity of a fluorophore-labeled protein remains constant upon protein-protein interaction. In this technique, a fluorophore-labeled protein is excited with vertically polarized light of an appropriate wavelength that selectively excites a subset of the fluorophores according to their relative orientation with the incoming beam. The resulting emission also has a directionality whose relationship in the vertical and horizontal planes defines anisotropy (r) as follows: r=(IVV-IVH)/(IVV+2IVH), where IVV and IVH are the fluorescence intensities of the vertical and horizontal components, respectively. Fluorescence anisotropy is sensitive to the rotational diffusion of a fluorophore, namely the apparent molecular size of a fluorophore attached to a protein, which is altered upon protein-protein interaction. In the present text, the use of fluorescence anisotropy as a tool to study protein-protein interactions was exemplified to address the binding between the protein mutated in the Shwachman-Diamond Syndrome (SBDS) and the Elongation factor like-1 GTPase (EFL1). Conventionally, labeling of a protein with a fluorophore is carried out on the thiol groups (cysteine) or in the amino groups (the N-terminal amine or lysine) of the protein. However, SBDS possesses several cysteines and lysines that did not allow site directed labeling of it. As an alternative technique, the dye 4&#39;,5&#39;-bis(1,3,2 dithioarsolan-2-yl) fluorescein was used to specifically label a tetracysteine motif, Cys-Cys-Pro-Gly-Cys-Cys, genetically engineered in the C-terminus of the recombinant SBDS protein. Fitting of the experimental data provided quantitative and mechanistic information on the binding mode between these proteins.

Protein interactions are at the heart of a cell&#39;s function. Calorimetric and spectroscopic techniques are commonly used to characterize them. Here we describe fluorescence anisotropy as a tool to study the interaction between the protein mutated in the Shwachman-Diamond Syndrome (SBDS) and the Elongation factor-like 1 GTPase (EFL1).

Anisotropia di fluorescenza come strumento per studiare le interazioni proteina-proteina

Protein-protein interactions play an essential role in the function of a living organism. Once an interaction has been identified and validated it is ...

Ricerca

Biochimica

Time-resolved ElectroSpray Ionization Hydrogen-deuterium Exchange Mass Spectrometry for Studying Protein Structure and Dynamics

Intrinsically disordered proteins (IDPs) have long been a challenge to structural biologists due to their lack of stable secondary structure elements. Hydrogen-Deuterium Exchange (HDX) measured at rapid time scales is uniquely suited to detect structures and hydrogen bonding networks that are briefly populated, allowing for the characterization of transient conformers in native ensembles. Coupling of HDX to mass spectrometry offers several key advantages, including high sensitivity, low sample consumption and no restriction on protein size. This technique has advanced greatly in the last several decades, including the ability to monitor HDX labeling times on the millisecond time scale. In addition, by incorporating the HDX workflow onto a microfluidic platform housing an acidic protease microreactor, we are able to localize dynamic properties at the peptide level. In this study, Time-Resolved ElectroSpray Ionization Mass Spectrometry (TRESI-MS) coupled to HDX was used to provide a detailed picture of residual structure in the tau protein, as well as the conformational shifts induced upon hyperphosphorylation.

Conformational flexibility plays a critical role in protein function. Herein, we describe the use of time-resolved electrospray ionization mass spectrometry coupled to hydrogen-deuterium exchange for probing the rapid structural changes that drive function in ordered and disordered proteins.

Scambio risolta in tempo ionizzazione elettrospray idrogeno-deuterio Spettrometria di Massa per lo studio delle proteine ​​Struttura e dinamica

Intrinsically disordered proteins (IDPs) have long been a challenge to structural biologists due to their lack of stable secondary structure elements. ...

Biotinylated Cell-penetrating Peptides to Study Intracellular Protein-protein Interactions

Here we present a protocol to study intracellular protein-protein interactions that is based on the widely used biotin-avidin pull-down system. The modification presented includes the combination of this technique with cell-penetrating sequences. We propose to design cell-penetrating baits that can be incubated with living cells instead of cell lysates and therefore the interactions found will reflect those that occur within the intracellular context. Connexin43 (Cx43), a protein that forms gap junction channels and hemichannels is down-regulated in high-grade gliomas. The Cx43 region comprising amino acids 266-283 is responsible for the inhibition of the oncogenic activity of c-Src in glioma cells. Here we use TAT as the cell-penetrating sequence, biotin as the pull-down tag and the region of Cx43 comprised between amino acids 266-283 as the target to find intracellular interactions in the hard-to-transfect human glioma stem cells. One of the limitations of the proposed method is that the molecule used as bait could fail to fold properly and, consequently, the interactions found could not be associated with the effect. However, this method can be especially interesting for the interactions involved in signal transduction pathways because they are usually carried out by intrinsically disordered regions and, therefore, they do not require an ordered folding. In addition, one of the advantages of the proposed method is that the relevance of each residue on the interaction can be easily studied. This is a modular system; therefore, other cell-penetrating sequences, other tags, and other intracellular targets can be employed. Finally, the scope of this protocol is far beyond protein-protein interaction because this system can be applied to other bioactive cargoes such as RNA sequences, nanoparticles, viruses or any molecule that can be transduced with cell-penetrating sequences and fused to pull-down tags to study their intracellular mechanism of action.

This is a protocol to study intracellular protein-protein interactions based on the biotin-avidin pull-down system with the novelty of combining cell-penetrating sequences. The main advantage is that the target sequence is incubated with living cells instead of cell lysates and therefore the interactions will occur within the cellular context.

Biotinylated Cell-penetrating peptidi per studiare le interazioni proteina-proteina intracellulare

Here we present a protocol to study intracellular protein-protein interactions that is based on the widely used biotin-avidin pull-down system. The ...

Using Three-color Single-molecule FRET to Study the Correlation of Protein Interactions

Single-molecule F&#246;rster resonance energy transfer (smFRET) has become a widely used biophysical technique to study the dynamics of biomolecules. For many molecular machines in a cell proteins have to act together&#160;with interaction partners in a functional cycle to fulfill their task. The extension of two-color to multi-color smFRET makes it possible to simultaneously probe more than one interaction or conformational change. This not only adds a new dimension to smFRET experiments but it also offers the unique possibility to directly study the sequence of events and to detect correlated interactions when using an immobilized sample and a total internal reflection fluorescence microscope (TIRFM). Therefore, multi-color smFRET is a versatile tool for studying biomolecular complexes in a quantitative manner and in a previously unachievable detail.
Here, we demonstrate how to overcome the special challenges of multi-color smFRET experiments on proteins. We present detailed protocols for obtaining the data and for extracting kinetic information. This includes trace selection criteria, state separation, and the recovery of state trajectories from the noisy data using a 3D ensemble Hidden Markov Model (HMM). Compared to other methods, the kinetic information is not recovered from dwell time histograms but directly from the HMM. The maximum likelihood framework allows us to critically evaluate the kinetic model and to provide meaningful uncertainties for the rates.
By applying our method to the heat shock protein 90 (Hsp90), we are able to disentangle the nucleotide binding and the global conformational changes of the protein. This allows us to directly observe the cooperativity between the two nucleotide binding pockets of the Hsp90 dimer.

Here, we present a protocol to obtain three-color smFRET data and its analysis with a 3D ensemble Hidden Markov Model. With this approach, scientists can extract kinetic information from complex protein systems, including cooperativity or correlated interactions.

Usando tre colori singola molecola FRET per studiare la correlazione delle interazioni della proteina

Single-molecule F&#246;rster resonance energy transfer (smFRET) has become a widely used biophysical technique to study the dynamics of biomolecules. For ...

Manipulating Living Cells to Construct Stable 3D Cellular Assembly Without Artificial Scaffold

Regenerative medicine and tissue engineering offer several advantages for the treatment of intractable diseases, and several studies have demonstrated the importance of 3-dimensional (3D) cellular assemblies in these fields. Artificial scaffolds have often been used to construct 3D cellular assemblies. However, the scaffolds used to construct cellular assemblies are sometimes toxic and may change the properties of the cells. Thus, it would be beneficial to establish a non-toxic method for facilitating cell-cell contact. In this paper, we introduce a novel method for constructing stable cellular assemblies by using optical tweezers with dextran. One of the advantages of this method is that it establishes stable cell-to-cell contact within a few minutes. This new method allows the construction of 3D cellular assemblies in a natural hydrophilic polymer and is expected to be useful for constructing next-generation 3D single-cell assemblies in the fields of regenerative medicine and tissue engineering.

We demonstrate a novel method for constructing a single-cell-based 3-dimensional (3D) assembly without an artificial scaffold.

Manipolazione delle cellule viventi per costruire assieme cellulare 3D stabile senza impalcatura artificiale

Regenerative medicine and tissue engineering offer several advantages for the treatment of intractable diseases, and several studies have demonstrated the ...

Large-scale Production of Recombinant RNAs on a Circular Scaffold Using a Viroid-derived System in Escherichia coli

With increasing interest in RNA biology and the use of RNA molecules in sophisticated biotechnological applications, the methods to produce large amounts of recombinant RNAs are limited. Here, we describe a protocol to produce large amounts of recombinant RNA in Escherichia coli based on co-expression of a chimeric molecule that contains the RNA of interest in a viroid scaffold and a plant tRNA ligase. Viroids are relatively small, non-coding, highly base-paired circular RNAs that are infectious to higher plants. The host plant tRNA ligase is an enzyme recruited by viroids that belong to the family Avsunviroidae, such as Eggplant latent viroid (ELVd), to mediate RNA circularization during viroid replication. Although ELVd does not replicate in E. coli, an ELVd precursor is efficiently transcribed by the E. coli RNA polymerase and processed by the embedded hammerhead ribozymes in bacterial cells, and the resulting monomers are circularized by the co-expressed tRNA ligase reaching a remarkable concentration. The insertion of an RNA of interest into the ELVd scaffold enables the production of tens of milligrams of the recombinant RNA per liter of bacterial culture in regular laboratory conditions. A main fraction of the RNA product is circular, a feature that facilitates the purification of the recombinant RNA to virtual homogeneity. In this protocol, a complementary DNA (cDNA) corresponding to the RNA of interest is inserted in a particular position of the ELVd cDNA in an expression plasmid that is used, along the plasmid to co-express eggplant tRNA ligase, to transform E. coli. Co-expression of both molecules under the control of strong constitutive promoters leads to production of large amounts of the recombinant RNA. The recombinant RNA can be extracted from the bacterial cells and separated from the bulk of bacterial RNAs taking advantage of its circularity.

Large-scale Production of Recombinant RNAs on a Circular Scaffold Using a Viroid-derived System in Escherichia coli

Here, we present a protocol to produce large amounts of recombinant RNA in Escherichia coli by co-expressing a chimeric RNA that contains the RNA of interest in a viroid scaffold and a plant tRNA ligase. The main product is a circular molecule that facilitates purification to homogeneity.

Produzione su larga scala di RNA ricombinante su un'impalcatura circolare utilizzando un sistema derivato Viroid in Escherichia coli

With increasing interest in RNA biology and the use of RNA molecules in sophisticated biotechnological applications, the methods to produce large amounts ...

A Fluorescence Fluctuation Spectroscopy Assay of Protein-Protein Interactions at Cell-Cell Contacts

A variety of biological processes involves cell-cell interactions, typically mediated by proteins that interact at the interface between neighboring cells. Of interest, only few assays are capable of specifically probing such interactions directly in living cells. Here, we present an assay to measure the binding of proteins expressed at the surfaces of neighboring cells, at cell-cell contacts. This assay consists of two steps: mixing of cells expressing the proteins of interest fused to different fluorescent proteins, followed by fluorescence fluctuation spectroscopy measurements at cell-cell contacts using a confocal laser scanning microscope. We demonstrate the feasibility of this assay in a biologically relevant context by measuring the interactions of the amyloid precursor-like protein 1 (APLP1) across cell-cell junctions. We provide detailed protocols on the data acquisition using fluorescence-based techniques (scanning fluorescence cross-correlation spectroscopy, cross-correlation number and brightness analysis) and the required instrument calibrations. Further, we discuss critical steps in the data analysis and how to identify and correct external, spurious signal variations, such as those due to photobleaching or cell movement.
In general, the presented assay is applicable to any homo- or heterotypic protein-protein interaction at cell-cell contacts, between cells of the same or different types and can be implemented on a commercial confocal laser scanning microscope. An important requirement is the stability of the system, which needs to be sufficient to probe diffusive dynamics of the proteins of interest over several minutes.

This protocol describes a fluorescence fluctuation spectroscopy-based approach to investigate interactions among proteins mediating cell-cell interactions, i.e. proteins localized in cell junctions, directly in living cells. We provide detailed guidelines on instrument calibration, data acquisition and analysis, including corrections to possible artefact sources.

Un'analisi di spettroscopia di fluorescenza fluttuazione delle interazioni proteina-proteina a contatti cellula-cellula

A variety of biological processes involves cell-cell interactions, typically mediated by proteins that interact at the interface between neighboring ...

Site-Directed Mutagenesis for In Vitro and In Vivo Experiments Exemplified with RNA Interactions in Escherichia Coli

Site-directed mutagenesis is a technique used to introduce specific mutations in DNA to investigate the interaction between small non-coding ribonucleic acid (sRNA) molecules and target messenger RNAs (mRNAs). In addition, site-directed mutagenesis is used to map specific protein binding sites to RNA. A 2-step and 3-step PCR based introduction of mutations is described. The approach is relevant to all protein-RNA and RNA-RNA interaction studies. In short, the technique relies on designing primers with the desired mutation(s), and through 2 or 3 steps of PCR synthesizing a PCR product with the mutation. The PCR product is then used for cloning. Here, we describe how to perform site-directed mutagenesis with both the 2- and 3-step approach to introduce mutations to the sRNA, McaS, and the mRNA, csgD, to investigate RNA-RNA and RNA-protein interactions. We apply this technique to investigate RNA interactions; however, the technique is applicable to all mutagenesis studies (e.g., DNA-protein interactions, amino-acid substitution/deletion/addition). It is possible to introduce any kind of mutation except for non-natural bases but the technique is only applicable if a PCR product can be used for downstream application (e.g., cloning and template for further PCR).

Site-Directed Mutagenesis for In Vitro and In Vivo Experiments Exemplified with RNA Interactions in Escherichia Coli

Site-directed mutagenesis is a technique used to introduce specific mutations in deoxyribonucleic acid (DNA). This protocol describes how to do site-directed mutagenesis with a 2-step and 3-step polymerase chain reaction (PCR) based approach, which is applicable to any DNA fragment of interest.

Mutagenesi sito-diretta per In Vitro e In Vivo esperimenti esemplificati con interazioni RNA in Escherichia Coli

Site-directed mutagenesis is a technique used to introduce specific mutations in DNA to investigate the interaction between small non-coding ribonucleic ...

Application of Biolayer Interferometry (BLI) for Studying Protein-Protein Interactions in Transcription

A transcription factor&#160;(TF) is a protein that regulates gene expression by interacting with the RNA polymerase, another TF, and/or template DNA. GrgA is a novel transcription activator found specifically in the obligate intracellular bacterial pathogen Chlamydia. Protein pulldown assays using affinity beads have revealed that GrgA binds two &#963; factors, namely &#963;66 and &#963;28, which recognize different sets of promoters for genes whose products are differentially required at developmental stages. We have used BLI to confirm and further characterize the interactions. BLI demonstrates several advantages over pulldown: 1) It reveals real-time association and dissociation between binding partners, 2) It generates quantitative kinetic parameters, and 3) It can detect bindings that pulldown assays often fail to detect. These characteristics have enabled us to deduce the physiological roles of GrgA in gene expression regulation in Chlamydia, and possible detailed interaction mechanisms. We envision that this relatively affordable technology can be extremely useful for studying transcription and other biological processes.

Application of Biolayer Interferometry BLI for Studying Protein-Protein Interactions in Transcription

Interactions of transcription factors (TFs) with the RNA polymerase are usually studied using pulldown assays. We apply a Biolayer Interferometry (BLI) technology to characterize the interaction of GrgA with the chlamydial RNA polymerase. Compared to pulldown assays, BLI detects real-time association and dissociation, offers higher sensitivity, and is highly quantitative.

Applicazione dell'interferometria biostrato (BLI) per lo studio delle interazioni tra proteine e proteine nella trascrizione

A transcription factor&#160;(TF) is a protein that regulates gene expression by interacting with the RNA polymerase, another TF, and/or template DNA. GrgA ...

Evaluation of Protein&#8211;Protein Interactions using an On-Membrane Digestion Technique

Numerous intracellular proteins physically interact in accordance with their intracellular and extracellular circumstances. Indeed, cellular functions largely depend on intracellular protein&#8211;protein interactions. Therefore, research regarding these interactions is indispensable to facilitating the understanding of physiologic processes. Co-precipitation of associated proteins, followed by mass spectrometry (MS) analysis, enables the identification of novel protein interactions. In this study, we have provided details of the novel technique of immunoprecipitation-liquid chromatography (LC)-MS/MS analysis combined with on-membrane digestion for the analysis of protein&#8211;protein interactions. This technique is suitable for crude immunoprecipitants and can improve the throughput of proteomic analyses. Tagged recombinant proteins were precipitated using specific antibodies; next, immunoprecipitants blotted onto polyvinylidene difluoride membrane pieces were subjected to reductive alkylation. Following trypsinization, the digested protein residues were analyzed using LC-MS/MS. Using this technique, we were able to identify several candidate associated proteins. Thus, this method is convenient and useful for the characterization of novel protein&#8211;protein interactions.

Here, we present a protocol for an on-membrane digestion technique for the preparation of samples for mass spectrometry. This technique facilitates the convenient analysis of protein&#8211;protein interactions.

Valutazione delle interazioni tra proteine e proteine utilizzando una tecnica di digestione On-Membrane

Numerous intracellular proteins physically interact in accordance with their intracellular and extracellular circumstances. Indeed, cellular functions ...