Gli autori vorrebbero riconoscere Chan-Sien Lay per fornire immagini di microcristalli purificati, Daniel Eriksson e Tom Caradoc-Davies per il supporto alla linea MX2 del Synchrotron australiano e Kathryn Flanagan e Andrew Fryga della struttura FlowCore presso la Monash University per la loro Un&#39;assistenza inestimabile.

<ol>
	<li>Tari, L. 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Questo protocollo fornisce due approcci per analizzare i microcristalli allo scopo di facilitare l&#39;analisi di cristalli molto piccoli che sarebbero stati trascurati in passato. Fasi critiche per la purificazione microcristallina Il protocollo presentato è stato ottimizzato utilizzando il polyhedrin Bombyx mori CPV1 espresso in cellule Sf9 come sistema di modello. Tuttavia, i microcristalli in vivo presentano una grande variabilità nella resistenza meccanica. Ad esempio, i cristalli ago-cathepsina B coltivati ​​nelle cellule insettuali sono rigidi e altamente resistenti a sollecitazioni meccaniche e possono essere purificate usando un protocollo simile a quello descritto qui 14 . D&#39;altra parte, i cristalli luciferasi a fiamma, anche coltivati ​​in cellule insettiche coltivate e con una simile morfologia ad ago, si dissolvono immediatamente sulla lisi cellulare 38 . Così, il protocollo di estrazione e purificazione di in vivoI cristalli dovranno essere adattati in una base di errori di prova per casi particolari come descritto nei passaggi 1.3, 2a.3-4, 2b.4, 3.1.3-4 e 3.2a.5. Per i cristalli fragili, la separazione (parziale) dai detriti cellulari potrebbe essere ottenuta mediante centrifugazione a bassa velocità ( cioè 200 xg) o aggiunta di un cuscino di saccarosio nella parte inferiore del tubo invece di ripetuta sonicazione. Come regola generale, i campioni di cristalli preparati per la raccolta dei dati di diffrazione non hanno bisogno di essere altamente purificati. Pertanto, la purificazione rapida è preferibile per raffreddare i cristalli prima che possano decadere. Applicazione a microcristalli in vitro e in vivo Anche se entrambi i protocolli sono qui illustrati con l&#39;analisi dei cristalli cresciuti in vivo , la metodologia può essere facilmente utilizzata per microcristalli coltivati ​​in vassoi di cristallo da proteine ​​ricombinanti purificate. In questoDovrebbero essere considerate le seguenti modifiche: i) il supporto della maglia può essere utilizzato per raccogliere i microcristalli direttamente dalla goccia di cristallizzazione piuttosto che trasferirli pipettando; Ii) poiché la quantità di cristalli può essere un fattore limitante, occorre prendere particolare attenzione quando si assorbe l&#39;eccesso di liquido dal supporto della maglia per evitare l&#39;eccessiva rimozione dei cristalli; Iii) devono essere testati diversi buffer di crioprotectant, come nella cristallografia convenzionale (vedi riferimento 30 ). D&#39;altra parte, per i cristalli in vivo , è fortemente raccomandato l&#39;approccio cellulare . Poiché le cellule sono più facili da visualizzare e manipolate rispetto ai cristalli purificati, questo approccio è più efficiente in termini di utilizzo di tempo e più accessibile ai ricercatori con poca o nessuna esperienza in microcristallografia. È anche più adatto per cristalli fragili che possono essere influenzati dall&#39;estrazione e pur<html> Ificazione dalla cella dovuta a cambiamenti nell&#39;ambiente chimico, diluizione delle proteine ​​circostanti e danni meccanici. Inoltre, le cellule offrono un ambiente protettivo ai cristalli, alleviando la necessità di aggiungere un crioprotettore. L&#39;emissione del trattamento di crioconservazione è stata precedentemente dimostrata per prevenire l&#39;accumulo di difetti e ha migliorato l&#39;isomorfismo tra singoli cristalli, per la raccolta di dati a temperatura ambiente 40 . Il protocollo cellulare inoltra anche l&#39;esigenza di incubazione in soluzione crioprotettori, consentendo comunque la raccolta dati a temperatura criogenica. Nel complesso, nella raccolta di dati di cellulo ha due vantaggi principali: 1) può produrre dati di migliore qualità e risoluzione più elevata per un determinato periodo di tempo di raggi 20 ; E 2) mantiene la proteina cristallizzata in un ambiente cellulare aumentando le possibilità di mantenere un ligando biologicamente rilevante. Ontent &quot;&gt; Limitazioni della tecnica Una complicazione intrinseca della microcristallografia seriale è l&#39;importante numero di set di dati parziali generati durante la raccolta dei dati. Di conseguenza, l&#39;elaborazione dei dati diventa un processo sempre più lungo. Tuttavia, il flusso di lavoro di base per l&#39;elaborazione dei dati può essere ampiamente automatizzato. Ad esempio, la scelta ottimale dei cristalli isomorfi può essere determinata mediante l&#39;analisi cluster gerarchica 28 , ad esempio realizzata in BLEND 36 . Inoltre, i programmi sviluppati per l&#39;analisi dei laser a elettroni liberi a raggi X (XFEL) possono anche essere adattati per elaborare i dati di microcristallografia seriali raccolti sulle linee di travi 2 di sincrotrone e, in alcuni rivelatori automatici di reti di maglie e di rilevamento di cristalli sono state implementate notevolmente facilitando la Raccolta dati 2 , 41 .

L&#39;uso della cristallografia a raggi X per la determinazione di strutture ad alta risoluzione di macromolecole biologiche ha registrato una costante progressione negli ultimi due decenni. La crescente assunzione di cristallografia a raggi X da ricercatori non esperti esemplifica la democratizzazione di questo approccio in molti settori della scienza della vita 1 . Storicamente, i cristalli con dimensioni inferiori a ~ 10 μm sono state considerate come impegnative, se non inutilizzabili, per la determinazione della struttura. La crescente disponibilità di microfocus dedicate a fonti di radiazione di sincrotrone in tutto il mondo e progressi tecnologici, come lo sviluppo di strumenti per la manipolazione dei microcristalli, hanno rimosso gran parte di queste barriere che hanno ostacolato l&#39;ampio utilizzo della microcristallografia a raggi X. Avanzamenti nella microcristallografia a raggi X seriali 2 , 3 e diffrazione a micro elettronica 4 haVe ha mostrato che l&#39;uso di micro- e nanocristalli per la determinazione della struttura non è solo possibile ma anche a volte preferibile all&#39;uso di grandi cristalli 5 , 6 , 7 . Questi progressi sono stati applicati per lo studio dei peptidi 8 e dei cristalli naturali prodotti dai virus degli insetti 9 , 10 . Sono ora utilizzati per una vasta gamma di macromolecole biologiche tra cui i sistemi più difficili come le proteine ​​a membrana e grandi complessi 11 . Per facilitare l&#39;analisi di questi microcristalli, sono stati analizzati in meso, proteine di membrana 12 in particolare e in chips microfluidica 13. La disponibilità di queste metodologie microcristallografia nuovi ha sollevato la possibilità di utilizzare inCristallizzazione vivo come un nuovo percorso per la biologia strutturale 14 , 15 , 16 che offre un&#39;alternativa alla cristallogenesi classica in vitro . Purtroppo, anche quando si possono produrre cristalli in vivo , rimangono diversi ostacoli quali la degradazione o la perdita di ligandi durante la depurazione dalle cellule, la difficoltà nella manipolazione e la visualizzazione dei cristalli alla radiazione di sincrotrone e negli esperimenti di diffusione a raggi X. Come cristalli alternativi sono stati analizzati anche direttamente all&#39;interno della cella senza alcuna fase di purificazione 17 , 18 , 19 . Un&#39;analisi comparativa suggerisce che tali approcci cellulari possono essere più efficienti dell&#39;analisi dei cristalli purificati e dei dati di resa di maggiore risoluzione 20 . Questo protocollo è inTendeva ad aiutare i ricercatori alla microcristallografia delle proteine. Fornisce metodologie che si concentrano sulla preparazione e manipolazione del campione per gli esperimenti di diffrazione a raggi X in un beamline di sincrotrone. Si propongono due opzioni utilizzando cristalli isolati per microcristallografia classica o cellule contenenti cristalli ordinate per citometria a flusso per l&#39;analisi cellulare ( Figura 1 ).

<table border="0" cellpadding="0" cellspacing="0" width="629">
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	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Sf9 cells</td>
			<td style="width:127px;">Life Technologies</td>
			<td style="width:119px;"></td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">SF900-SFM insect medium</td>
			<td style="width:127px;">Life Technologies</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">1L cell culture flask</td>
			<td style="width:127px;">Thermofisher Scientific</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Shaking incubator for insect cell culture</td>
			<td style="width:127px;">Eppendorf</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">50mL conical tubes</td>
			<td style="width:127px;">Falcon</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Centrifuge with swing buckets for 50mL tubes</td>
			<td style="width:127px;">Eppendorf</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Sonicator equiped with a 19mm probe</td>
			<td>MSE Soniprep 150&#160;</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Glass slides</td>
			<td style="width:127px;">Hampton Research</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Hemacytometer</td>
			<td style="width:127px;">Sigma-Aldrich</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Propidium iodide</td>
			<td style="width:127px;">Thermofisher Scientific</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">BD Influx cell sorter&#160;</td>
			<td style="width:127px;">BD Biosciences</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Hampton Heavy atom screens</td>
			<td style="width:127px;">Hampton Research</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Microcentrifuge</td>
			<td style="width:127px;">Eppendorf</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="105">
			<td height="105" style="height:105px;width:216px;">Micromesh</td>
			<td style="width:127px;">Mitigen</td>
			<td style="width:119px;"></td>
			<td style="width:167px;">700/25 meshes offer a larger surface. Indexed meshes can be purchased for systematic studies.</td>
		</tr>
		<tr height="147">
			<td height="147" style="height:147px;width:216px;">Paper wick</td>
			<td style="width:127px;">Mitigen</td>
			<td style="width:119px;"></td>
			<td style="width:167px;">The size of the paper wick can be varied for optimal flow. This will largely depend on the nature of the crystals and cryoprotectant used.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Ethylene glycol</td>
			<td style="width:127px;">Sigma-Aldrich</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Trypan blue</td>
			<td style="width:127px;">Life Technologies</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="231">
			<td height="231" style="height:231px;width:216px;">MX2 microfocus beamline</td>
			<td style="width:127px;">Australian Synchrotron</td>
			<td style="width:119px;"></td>
			<td style="width:167px;">A list of available microfocus beamlines can be found in Boudes et al. (2014) Reflections on the Many Facets of Protein 
			Microcrystallography. Australian Journal of Chemistry 67 (12), 1793&#8211;1806, 
			doi:10.1071/CH14455.</td>
		</tr>
	</tbody>
</table>

microcrystallography of protein crystals and in cellulo diffraction

L&#39;avvento di linee di microfocus di alta qualità in molti impianti di sincrotrone ha consentito l&#39;analisi di routine di cristalli inferiori a 10 μm nella loro dimensione più grande, che rappresentavano una sfida. Presentiamo due flussi di lavoro alternativi per la determinazione della struttura di microcristalli proteici mediante cristallografia a raggi X con particolare attenzione ai cristalli in vivo . I microcristalli vengono estratti dalle cellule per ultrasuoni e purificati mediante centrifugazione differenziale o analizzati in cellulo dopo la selezione delle cellule mediante citometria a flusso di cellule contenenti cristalli. Opzionalmente, cristalli purificati o cellule contenenti cristalli vengono imbevuti in soluzioni di atomi pesanti per la fase di sperimentazione. Questi campioni vengono quindi preparati per esperimenti di diffrazione in modo analogo mediante applicazione su un supporto micromesh e raffreddamento a flash in azoto liquido. Breve descrizione e confronto di esperimenti di diffrazione seriale di microcristalli isolati e di cristallo-Contenenti le cellule utilizzando un beamline di sincrotrino microfocus per produrre set di dati adatti per la fase di fasi, la costruzione di modelli e la raffinatezza. Questi flussi di lavoro sono esemplificati con i cristalli del polyhedrin Bombyx mori cypovirus 1 (BmCPV1) prodotto dall&#39;infezione di cellule insettiche con un baculovirus ricombinante. In questo caso, l&#39;analisi cellulare è più efficace dell&#39;analisi dei cristalli purificati e produce una struttura in ~ 8 giorni dall&#39;espressione al raffinamento.

Viene presentata una panoramica di entrambi i metodi alternativi per la determinazione della struttura utilizzando microcristalli in vivo ( Figura 1 ). La polihedra può essere facilmente purificata mediante sonicazione e centrifugazione. A causa della loro densità, formano uno strato nella parte inferiore del tubo sotto uno strato di detriti che possono essere rimossi pipettando ( Figura 3a e 3b ). Il campione viene quindi sottoposto a diversi cicli di sonicazione e lavaggi fino a raggiungere un livello sufficiente di purezza come giudicato dall&#39;aspetto bianco e gessoso del pellet ( Figura 3c ). Per l&#39;approccio in cellulo , il profilo di citometria di flusso di cellule non infette viene confrontato con il profilo di cellule contenenti cristalli e utilizzato per determinare quale popolazione cellulare deve essere selezionata per ordinare le cellule contenenti cristalli lontano daLe altre cellule ( Figura 4 ). In questo esempio, le celle contenenti poliedri hanno una dispersione laterale superiore rispetto alle cellule non infette. Altre differenze nei modelli di dispersione possono essere osservate per altri tipi di cristalli e il gating deve essere modificato di conseguenza. I microcristalli purificati o le cellule contenenti cristalli vengono pipettate su un micromesh ( Figura 5 ). Le cellule colorate con blu di trypan possono essere facilmente visualizzate usando telecamere disponibili alla maggior parte delle linee di sincrotrone ( Figura 5b ), mentre i cristalli purificati sono laboriosi per identificare e allinearsi con il raggio a raggi X a causa della loro piccola dimensione ( Figura 5c ). Quando si raccolgono i dati, è meglio procedere in un modello di griglia al fine di ridurre al minimo le procedure di centraggio e per evitare di esporre due volte la stessa cella o cristallo, o mancanti ( Figura 5a ). Raccolta datiDevono essere trattati con attenzione per tenere conto delle differenze nei limiti di diffrazione, dell&#39;effetto dei danni da radiazioni e di una possibile mancanza di isomorfismo. <img alt="Figura 1" class="xfigimg" src="/files/ftp_upload/55793/55793fig1.jpg" /> Figura 1 : Panoramica di due metodi per la determinazione della struttura usando microcristalli in vivo . I microcristalli vengono prodotti in coltura cellulare mediante sovraespressione della proteina di interesse utilizzando un sistema di espressione ricombinante. In casi specifici, i microcristalli possono anche essere prodotti naturalmente dalle cellule in condizioni particolari. La fila superiore mostra l&#39;approccio classico in cui le cellule sono lisate ei cristalli vengono purificati mediante centrifugazione differenziale. I microcristalli purificati vengono pipettati su un micromesh. Dopo la rimozione del liquido in eccesso, si aggiunge una soluzione di crioprotectant, si rimuove il liquido in eccesso e la maglia viene raffreddata a flash. Per raggi X dif<html> Gli esperimenti di frazione, i cristalli sono accuratamente allineati con il raggio a raggi X, che può essere il passo di limitazione della velocità nella raccolta dei dati. La riga inferiore presenta l&#39;approccio cellulare . Le cellule sono ordinate per citometria a flusso per selezionare specificamente le cellule contenenti cristalli. Le cellule sono macchiate con blu tripanico per facilitare la visualizzazione e diffondere su un micromesh. In questo caso, l&#39;aggiunta di un crioprotectante è facoltativa. Per le esperienze di diffrazione dei raggi X, le cellule sono facilmente visualizzate usando tipiche telecamere disponibili nelle fasce di sincrotrone che facilitano il processo di centraggio. Immagine di sistema automatico di cromatografia liquida a cura di GE Healthcare AB, Uppsala, Svezia. <a href="https://ecsource-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/55793/55793fig1large.jpg" target="_blank">Clicca qui per visualizzare una versione più grande di questa figura.</a> <img alt="figura 2" class="xfigimg" src="/files/ftp_upload/55793/55793fig2.jpg" /> = &quot;Xfig&quot;&gt; Figura 2 : Esempi di microcristalli in vivo . Esempi selezionati di cristalli in vivo: ( a ) le cellule Sf9 con poliedra di cypovirus; ( B ) le cellule LD652 con sferoidi di entomopoxvirus; C ) Bacillus thuringiensis con cristalli Cry3A (freccia); ( D ) le cellule Sf9 con i cristalli Trypanosoma brucei cathepsin B; ( E ) le cellule Sf21 con cristalli calcineurin (frecce); ( F ) monoslocce COS7 con PKA: cristalli Inka1; ( G ) le cellule CHO con cristalli IgG. Riprodotto da 14 , 18 , 29 , 37 , 39 , 42 , con autorizzazione. Barra di scala = 10 μm. <a href="https://ecsource-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/55793/55793fig2large.jpg" target="_blank">Clicca qui per vedere un largo</a>Er di questa figura. <img alt="Tabella 1" src="/files/ftp_upload/55793/55793table1.jpg" /> Tabella 1: Sintesi dei cristalli in vivo cresciuti in cellule coltivate che esprimono la proteina ricombinante. <a href="https://ecsource-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/55793/55793table1large.jpg" target="_blank">Clicca qui per visualizzare una versione più grande di questa figura.</a>

Watch this Scientific Journal Video about Microcrystallography of Protein Crystals and In Cellulo Diffraction at JoVE.com

Microcrystallography of Protein Crystals and In Cellulo Diffraction

Viene presentato un protocollo per la cristallografia a raggi X utilizzando microcristalli di proteine. Sono stati confrontati due esempi che analizzano microcristalli in vivo dopo la purificazione o in cellulo .

Microcristallografia dei cristalli proteici e<em&gt; In Cellulo</em&gt; Diffrazione

The advent of high-quality microfocus beamlines at many synchrotron facilities has permitted the routine analysis of crystals smaller than 10 &#181;m in ...

microcrystallography-of-protein-crystals-and-in-cellulo-diffraction

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Biochemistry

Microcrystallography of Protein Crystals and In Cellulo Diffraction

9.0K Views.  Monash University.  Viene presentato un protocollo per la cristallografia a raggi X utilizzando microcristalli di proteine. Sono stati confrontati due esempi che analizzano microcristalli in vivo dopo la purificazione o in cellulo . 

Video: Microcrystallography of Protein Crystals and In Cellulo Diffraction

Visualization of Protein-protein Interaction in Nuclear and Cytoplasmic Fractions by Co-immunoprecipitation and In Situ Proximity Ligation Assay

Protein-protein interactions are involved in thousands of cellular processes and occur in distinct spatial context. Traditionally, co-immunoprecipitation is a popular technique to detect protein-protein interactions. Subsequent Western blot analysis is the most common method to visualize co-immunoprecipitated proteins. Recently, the proximity ligation assay has become a powerful tool to visualize protein-protein interactions in situ and provides the possibility to quantify protein-protein interactions by this method. Similar to conventional immunocytochemistry, the proximity ligation assay technique is also based on the accessibility of primary antibodies to the antigens, but in contrast, proximity ligation assay detects protein-protein interactions with a unique technique involving rolling-circle PCR, while conventional immunocytochemistry only shows co-localization of proteins.
Nuclear factor 90 (NF90) and RNA-binding motif protein 3 (RBM3) have been previously demonstrated as interacting partners. They are predominantly localized in the nucleus, but also migrate into the cytoplasm and regulate signaling pathways in the cytoplasmic compartment. Here, we compared NF90-RBM3 interaction in both the nucleus and the cytoplasm by co-immunoprecipitation and proximity ligation assay. In addition, we discussed the advantages and limitations of these two techniques in visualizing protein-protein interactions in respect to spatial distribution and the properties of protein-protein interactions.

Visualization of Protein-protein Interaction in Nuclear and Cytoplasmic Fractions by Co-immunoprecipitation and In Situ Proximity Ligation Assay

Protein-protein interactions can occur in both the nucleus and the cytoplasm of a cell. To investigate these interactions, traditional co-immunoprecipitation and modern proximity ligation assay are applied. In this study, we compare these two methods to visualize the distribution of NF90-RBM3 interactions in the nucleus and the cytoplasm.

Visualizzazione di interazione proteina-proteina in nucleare e citoplasmatica frazioni da Co-immunoprecipitazione e<em&gt; In Situ</em&gt; Proximity Assay legatura

Protein-protein interactions are involved in thousands of cellular processes and occur in distinct spatial context. Traditionally, co-immunoprecipitation ...

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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. ...

Growing Protein Crystals with Distinct Dimensions Using Automated Crystallization Coupled with In Situ Dynamic Light Scattering

The automated crystallization device is a patented technique1 especially developed for monitoring protein crystallization experiments with the aim to precisely maneuver the nucleation and crystal growth towards desired sizes of protein crystals. The controlled crystallization is based on sample investigation with in situ Dynamic Light Scattering (DLS) while all visual changes in the droplet are monitored online with the help of a microscope coupled to a CCD camera, thus enabling a full investigation of the protein droplet during all stages of crystallization. The use of in situ DLS measurements throughout the entire experiment allows a precise identification of the highly supersaturated protein solution transitioning to a new phase &#8211; the formation of crystal nuclei. By identifying the protein nucleation stage, the crystallization can be optimized from large protein crystals to the production of protein microcrystals. The experimental protocol shows an interactive crystallization approach based on precise automated steps such as precipitant addition, water evaporation for inducing high supersaturation, and sample dilution for slowing induced homogeneous nucleation or reversing phase transitions.

Growing Protein Crystals with Distinct Dimensions Using Automated Crystallization Coupled with In Situ Dynamic Light Scattering

Here we present a protocol for controlled production of protein microcrystals. The process uses an automated device allowing controlled manipulation of several crystallization parameters. The protein crystallization is carried-out by controlled and automated addition of crystallization solutions while monitoring and investigating the radius distribution of particles in the crystallization droplet.

Cristalli della proteina crescente con dimensioni distinte utilizzando automatizzato cristallizzazione accoppiato con In Situ dispersione della luce dinamica

The automated crystallization device is a patented technique1 especially developed for monitoring protein crystallization experiments with the aim to ...

2 in 1: One-step Affinity Purification for the Parallel Analysis of Protein-Protein and Protein-Metabolite Complexes

Cellular processes are regulated by interactions between biological molecules such as proteins, metabolites, and nucleic acids. While the investigation of protein-protein interactions (PPI) is no novelty, experimental approaches aiming to characterize endogenous protein-metabolite interactions (PMI) constitute a rather recent development. Herein, we present a protocol that allows simultaneous characterization of the PPI and PMI of a protein of choice, referred to as bait. Our protocol was optimized for Arabidopsis cell cultures and combines affinity purification (AP) with mass spectrometry (MS)-based protein and metabolite detection. In short, transgenic Arabidopsis lines, expressing bait protein fused to an affinity tag, are first lysed to obtain a native cellular extract. Anti-tag antibodies are used to pull down protein and metabolite partners of the bait protein. The affinity-purified complexes are extracted using a one-step methyl tert-butyl ether (MTBE)/methanol/water method. Whilst metabolites separate into either the polar or the hydrophobic phase, proteins can be found in the pellet. Both metabolites and proteins are then analyzed by ultra-performance liquid chromatography-mass spectrometry (UPLC-MS or UPLC-MS/MS). Empty-vector (EV) control lines are used to exclude false positives. The major advantage of our protocol is that it enables identification of protein and metabolite partners of a target protein in parallel in near-physiological conditions (cellular lysate). The presented method is straightforward, fast, and can be easily adapted to biological systems other than plant cell cultures.

Protein-protein and protein-metabolite interactions are crucial for all cellular functions. Herein, we describe a protocol that allows parallel analysis of these interactions with a protein of choice. Our protocol was optimized for plant cell cultures and combines affinity purification with mass spectrometry-based protein and metabolite detection.

2 in 1: purificazione di affinità per l'analisi parallela di proteina-proteina e proteina-metabolita complessi in un unico passaggio

Cellular processes are regulated by interactions between biological molecules such as proteins, metabolites, and nucleic acids. While the investigation of ...

Measuring and Interpreting Oxygen Consumption Rates in Whole Fly Head Segments

Regulated metabolic activity is essential for the normal functioning of living cells. Indeed, altered metabolic activity is causally linked with the progression of cancer, diabetes, neurodegeneration, and aging to name a few. For instance, changes in mitochondrial activity, the cell&#39;s metabolic powerhouse, have been characterized in many such diseases. Generally, the oxygen consumption rates of mitochondria were considered a reliable readout of mitochondrial activity and measurements in some of these studies were based on isolated mitochondria or cells. However, such conditions may not represent the complexity of a whole tissue. Recently, we have developed a novel method that enables the dynamic measurement of oxygen consumption rates from whole isolated fly heads. By utilizing this method, we have recorded lower oxygen consumption rates of the whole head segment in young versus aged flies. Secondly, we have discovered that lysine deacetylase inhibitors rapidly alter the oxygen consumption in the whole head. Our novel technique may therefore aid in uncovering new properties of various drugs, which may impact metabolic rates. Furthermore, our method may give a better understanding of metabolic behavior in an experimental setup that more closely resembles physiological states.

Measuring alterations in metabolic rates is central to understanding the progression of various diseases and aging. Here, we present a novel technique to measure whole head oxygen consumption that more closely resembles the physiological state and may aid in revealing novel drugs that modify mitochondrial activity.

Misurare e interpretare i tassi di consumo di ossigeno in tutto Vola testa segmenti

Regulated metabolic activity is essential for the normal functioning of living cells. Indeed, altered metabolic activity is causally linked with the ...

High Sensitivity Measurement of Transcription Factor-DNA Binding Affinities by Competitive Titration Using Fluorescence Microscopy

Accurate quantification of transcription factor (TF)-DNA interactions is essential for understanding the regulation of gene expression. Since existing approaches suffer from significant limitations, we have developed a new method for determining TF-DNA binding affinities with high sensitivity on a large scale. The assay relies on the established fluorescence anisotropy (FA) principle but introduces important technical improvements. First, we measure a full FA competitive titration curve in a single well by incorporating TF and a fluorescently labeled reference DNA in a porous agarose gel matrix. Unlabeled DNA oligomer is loaded on the top as a competitor and, through diffusion, forms a spatio-temporal gradient. The resulting FA gradient is then read out using a customized epifluorescence microscope setup. This improved setup greatly increases the sensitivity of FA signal detection, allowing both weak and strong binding to be reliably quantified, even for molecules of similar molecular weights. In this fashion, we can measure one titration curve per well of a multi-well plate, and through a fitting procedure, we can extract both the absolute dissociation constant (KD) and active protein concentration. By testing all single-point mutation variants of a given consensus binding sequence, we can survey the entire binding specificity landscape of a TF, typically on a single plate. The resulting position weight matrices (PWMs) outperform those derived from other methods in predicting in vivo TF occupancy. Here, we present a detailed guide for implementing HiP-FA on a conventional automated fluorescent microscope and the data analysis pipeline.

Here we present a novel method for determining binding affinities at equilibrium and in solution with high sensitivity on a large scale. This improves the quantitative analysis of transcription factor-DNA binding. The method is based on automated fluorescence anisotropy measurements in a controlled delivery system.

Alta sensibilità misura dell'affinità di legame di DNA di fattore di trascrizione per titolazione competitivo mediante microscopia a fluorescenza

Accurate quantification of transcription factor (TF)-DNA interactions is essential for understanding the regulation of gene expression. Since existing ...

Analysis of Protein Folding, Transport, and Degradation in Living Cells by Radioactive Pulse Chase

Radioactive pulse-chase labeling is a powerful tool for studying the conformational maturation, the transport to their functional cellular location, and the degradation of target proteins in live cells. By using short (pulse) radiolabeling times (&lt;30 min) and tightly controlled chase times, it is possible to label only a small fraction of the total protein pool and follow its folding. When combined with nonreducing/reducing SDS-polyacrylamide gel electrophoresis (SDS-PAGE) and immunoprecipitation with (conformation-specific) antibodies, folding processes can be examined in great detail. This system has been used to analyze the folding of proteins with a huge variation in properties such as soluble proteins, single and multi-pass transmembrane proteins, heavily N- and O-glycosylated proteins, and proteins with and without extensive disulfide bonding. Pulse-chase methods are the basis of kinetic studies into a range of additional features, including co- and posttranslational modifications, oligomerization, and polymerization, essentially allowing the analysis of a protein from birth to death. Pulse-chase studies on protein folding are complementary with other biochemical and biophysical methods for studying proteins in vitro by providing increased temporal resolution and physiological information. The methods as described within this paper are adapted easily to study the folding of almost any protein that can be expressed in mammalian or insect-cell systems.

Here we describe a protocol for a general pulse-chase method that allows the kinetic analysis of folding, transport, and degradation of proteins to be followed in live cells.

Analisi della piegatura della proteina, trasporto e degradazione in cellule viventi di radioattivo Pulse Chase

Radioactive pulse-chase labeling is a powerful tool for studying the conformational maturation, the transport to their functional cellular location, and ...

Fully Autonomous Characterization and Data Collection from Crystals of Biological Macromolecules

High-brilliance X-ray beams coupled with automation have&#160;led to the use of synchrotron-based macromolecular X-ray crystallography (MX) beamlines for even the most challenging projects in structural biology. However, most facilities still require the presence of a scientist on site to perform the experiments. A new generation of automated beamlines dedicated to the fully automatic characterization of, and data collection from, crystals of biological macromolecules has recently been developed. These beamlines represent a new tool for structural biologists to screen the results of initial crystallization trials and/or the collection of large numbers of diffraction data sets, without users having to control the beamline themselves. Here we show how to set up an experiment for automatic screening and data collection, how an experiment is performed at the beamline, how the resulting data sets are processed, and how, when possible, the crystal structure of the biological macromolecule is solved.

Here, we describe how to use the automated screening and data collection options available at some synchrotron beamlines. Scientists send cryocooled samples to the synchrotron, and the diffraction properties are screened, the data sets are collected and processed and, where possible, a structure solution is carried out&#8212;all without human intervention.

Raccolta di dati dai cristalli delle macromolecole biologiche e completamente autonoma caratterizzazione

High-brilliance X-ray beams coupled with automation have&#160;led to the use of synchrotron-based macromolecular X-ray crystallography (MX) beamlines for ...

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 ...

Creating Highly Specific Chemically Induced Protein Dimerization Systems by Stepwise Phage Selection of a Combinatorial Single-Domain Antibody Library

Protein dimerization events that occur only in the presence of a small-molecule ligand enable the development of small-molecule biosensors for the dissection and manipulation of biological pathways. Currently, only a limited number of chemically induced dimerization (CID) systems exist and engineering new ones with desired sensitivity and selectivity for specific small-molecule ligands remains a challenge in the field of protein engineering. We here describe a high throughput screening method, combinatorial binders-enabled selection of CID (COMBINES-CID), for the de novo engineering of CID systems applicable to a large variety of ligands. This method uses the two-step selection of a phage-displayed combinatorial nanobody library to obtain 1) &#34;anchor binders&#34; that first bind to a ligand of interest and then 2) &#34;dimerization binders&#34; that only bind to anchor binder-ligand complexes. To select anchor binders, a combinatorial library of over 109 complementarity-determining region (CDR)-randomized nanobodies is screened with a biotinylated ligand and hits are validated with the unlabeled ligand by bio-layer interferometry (BLI). To obtain dimerization binders, the nanobody library is screened with anchor binder-ligand complexes as targets for positive screening and the unbound anchor binders for negative screening. COMBINES-CID is broadly applicable to select CID binders with other immunoglobulin, non-immunoglobulin, or computationally designed scaffolds to create biosensors for in vitro and in vivo detection of drugs, metabolites, signaling molecules, etc.

Creating chemically induced protein dimerization systems with desired affinity and specificity for any given small molecule ligand would have many biological sensing and actuation applications. Here, we describe an efficient, generalizable method for de novo engineering of chemically induced dimerization systems via the stepwise selection of a phage-displayed combinatorial single-domain antibody library.

Creazione di sistemi di dimerizzazione delle proteine altamente specifici indotta chimicamente mediante la selezione stepwise di phage di una biblioteca combinatoria di anticorpi a dominio singolo