Diese Arbeit wurde durch Zuschüsse aus Consejo Nacional de Ciencias y Tecnología-México (CONACyT-Mexico) (82.614, 153.998, 253.549 und 178.359 in Investigación Científica Básica sowie 118 in Investigación en Fronteras de la Ciencia) und von Dirección General de unterstützt Asuntos de Personal Académico-Universidad Nacional Autónoma de México (UNAM DGAPA-; PAPIIT IN202616, IN203612).

<ol>
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Die Autoren haben nichts zu offenbaren.

In den letzten 20 Jahren haben die Genome vieler Organismen sequenziert worden, und obwohl eine Fülle von Daten Genomsequenz generiert wurde, ist funktionale Auslegung Rückstand und damit behindert unser Verständnis der Genomfunktion. Genfunktionen in Genomen basieren häufig auf Ähnlichkeit mit Genen bekannter Funktion oder Auftreten von konservierten Motive zugeordnet. Jedoch ist die genaue Funktion eines bestimmten Gens häufig nicht bekannt. Insbesondere vorhergesagte strukturelle Gene für Enzyme können nicht ...

Glycerol-basierten Lipiden wie Triglyceriden und (glycero) Phospholipide sind wichtige und wohl die bekanntesten Lipidklassen 1. Triacylglycerolen (TAGs) sind Fette oder Öle, die üblicherweise als Speicherlipiden funktionieren und daher als potentielle Energie und Kohlenstoffquellen. TAGs können durch Lipasen abgebaut werden, die häufig durch den produzierenden Organismus ausgeschieden werden externe TAGs zu verdauen und sie als Kohlenstoffquellen zur Verfügung zu stellen. Auch wurden Lipasen weit über die Jahre studiert aufgrund ihrer wichtigen biotechnologischen Anwendungen 2. Auf Grund ihrer amphiphilen Natur und ihrer nahezu zylindrischen Form, (glycero) Phospholipide weisen membranbildenden Eigenschaften und in der Regel die Hauptlipidkomponenten einer doppelschichtigen Membran 3 bilden. In einfachen Mikroorganismen wie dem Bakterium Escherichia coli, nur drei Hauptkopfgruppe Varianten, Phosphatidylglycerin (PG), Cardiolipin (CL) und phosphatidylethanolamine (PE) angetroffen werden , obwohl man sich bewusst sein sollte , dass jeder von ihnen kann 4 mit einer beträchtlichen Anzahl unterschiedlicher Fettacylketten an der sn - 1 oder sn - 2 - Position, die zu einer großen Anzahl von verschiedenen molekularen Spezies ersetzt werden . Andere Bakterien könnten haben andere Phospholipide zusätzlich oder statt. Zum Beispiel Sinorhizobium meliloti, ein Bodenbakterium, das mit der Leguminosen Luzerne (Medicago sativa) , um eine Stickstoff-fixierenden Knöllchen Symbiose zu bilden, enthält zusätzlich zu PE ein zweites zwitterionisches Phospholipid, Phosphatidylcholin (PC) 5 in der Lage ist. Auch Lipide nicht, die Phosphor oder Glycerin können amphiphile und bilden einen Teil der Zellmembran sein. Beispielsweise von Phosphor beschränkende Wachstumsbedingungen in S. meliloti, (glycero) Phospholipide sind weitgehend durch Membranlipide ersetzt , die nicht Phosphor enthalten, dh Sulfolipide, Ornithin Lipide und diacylglyceryl trimethylhomoserine (DGTS) 6. In Bakterien ist DGTS von Diacylglycerin (DAG) in einem zweistufigen Weg gebildet 7 , aber die Quelle für DAG Generation war nicht klar. Pulse-Chase - Experimente vorgeschlagen , dass PC ein Vorläufer für 8 DGTS sein könnte und mit Hilfe der Methodik in diesem Manuskript beschrieben wir eine Phospholipase C identifizieren konnte (PLCP, SMc00171) , die unter Phosphor einschränkenden Bedingungen gebildet wird , und die PC in DAG umwandeln und Phosphocholin 8. In einer separaten Studie entdeckten wir , dass eine Acyl-CoA - Synthetase (FADD) defizienten Mutante von S. meliloti oder von Escherichia coli freien Fettsäuren akkumuliert , wenn 9 stationären Wachstumsphase eintritt. Obwohl diese Fettsäuren schienen von Membranlipiden abgeleitet werden, die genaue Quelle für die freien Fettsäuren oder die Enzym (e) zu befreien, sie waren nicht bekannt. Auch in diesem Manuskript die Strategie skizziert den Einsatz, zwei Patatin-like 10 (phospho) Lipasen (SMc00930 und SMc01003), die zur Bildung von freien Fettsäuren in S. beigetragen meliloti 11 vorhergesagt wurden. Überraschenderweise verwendet SMc01003 DAG als Substrat Umwandlung in Monoacylglycerin und schließlich 11 Glycerin und freien Fettsäuren. Daher ist SMc01003 eine DAG-Lipase (DGLA). Obwohl eine Reihe von Algorithmen zur Vorhersage Potential (phospho) sind Lipasen 12,13, deren genaue Funktion und physiologische Rolle ist in der Regel nicht bekannt. Hier beschreiben wir ein Protokoll, zu klonen und überexprimiert vorhergesagt oder Potential (phospho) Lipasen. Diese Handschrift wird erläutert, wie Enzymassays können unter Verwendung von künstlichen chromogene Substrate für die überexprimiert (phospho) Lipase entwickelt und optimiert werden. Wir liefern Beispiele, wie mit einem optimierten Enzymtest die reale (phospho) Lipasesubstrats begegnet werden kann und wie diese Erkenntnisse könnten unser Verständnis der mikrobiellen Physiologie bereichern.

<table><tbody><tr><td>Chloroform</td><td>JT Baker</td><td>9180-03</td><td>TLC analysis &amp;amp; Lipid extraction</td></tr><tr><td>Methanol</td><td>JT Baker</td><td>9070-03</td><td>TLC analysis &amp;amp; Lipid extraction</td></tr><tr><td>Acetic Acid</td><td>JT Baker</td><td>9507-05</td><td>TLC analysis &amp;amp; Lipid extraction</td></tr><tr><td>Hexanes</td><td>JT Baker</td><td>9309-02</td><td>TLC analysis &amp;amp; Lipid extraction</td></tr><tr><td>Diethylether</td><td>Sigma</td><td>32203</td><td>Enzymatic assays</td></tr><tr><td>bidistilled water</td><td>&amp;nbsp;ANY</td><td>&amp;nbsp;NA</td><td>Enzymatic assays</td></tr><tr><td>Tris Base</td><td>Sigma</td><td>T-1503</td><td>Enzymatic assays</td></tr><tr><td>HCl</td><td>Baker</td><td>9535-02</td><td>Enzymatic assays</td></tr><tr><td>NaCl</td><td>Baker</td><td>3624-01</td><td>Enzymatic assays</td></tr><tr><td>Triton X-100</td><td>Sigma</td><td>X-100</td><td>Enzymatic assays</td></tr><tr><td>LB broth</td><td>ANY</td><td>NA</td><td>Bacterial growth, 10 g tryptone + 5 g yeast extract + 10 g NaCl per liter of bidistilled water</td></tr><tr><td>tryptone</td><td>Becton Dickinson and Company</td><td>211705</td><td>Bacterial growth</td></tr><tr><td>yeast extract</td><td>Becton Dickinson and Company</td><td>212750</td><td>Bacterial growth</td></tr><tr><td>TY broth</td><td>ANY</td><td>NA</td><td>Bacterial growth, 8 g tryptone + 3 g yeast extract + 66 mg CaCl&lt;sub&gt;2&lt;/sub&gt;&amp;#183;2H&lt;sub&gt;2&lt;/sub&gt;O per liter of bidistilled water</td></tr><tr><td>CaCl&lt;sub&gt;2&lt;/sub&gt;&amp;#183;2H&lt;sub&gt;2&lt;/sub&gt;O</td><td>Baker</td><td>1332-01</td><td>Enzymatic assays</td></tr><tr><td>isopropyl-&amp;beta;-D-thiogalactoside (IPTG)</td><td>Invitrogen</td><td>15529-019</td><td>Bacterial growth</td></tr><tr><td>Diethanolamine</td><td>Sigma</td><td>D-8885</td><td>Enzymatic assays</td></tr><tr><td>MnCl&lt;sub&gt;2&lt;/sub&gt;</td><td>Sigma</td><td>221279</td><td>Enzymatic assays</td></tr><tr><td>Phospholipase A&lt;sub&gt;2&lt;/sub&gt; snake venom</td><td>Sigma</td><td>P0790</td><td>Enzymatic assays</td></tr><tr><td>Phospholipase C &lt;em&gt;Clostridium perfringens&lt;/em&gt;</td><td>Sigma</td><td>P7633</td><td>Enzymatic assays</td></tr><tr><td>Bis-&lt;em&gt;p&lt;/em&gt;-nitrophenyl phosphate</td><td>Sigma</td><td>07422AH</td><td>Enzymatic assays</td></tr><tr><td>&lt;em&gt;p&lt;/em&gt;-nitrophenyl stearate</td><td>Sigma</td><td>N3627</td><td>Enzymatic assays</td></tr><tr><td>&lt;em&gt;p&lt;/em&gt;-nitrophenyl dodecanoate</td><td>Sigma</td><td>61716</td><td>Enzymatic assays</td></tr><tr><td>&lt;em&gt;p&lt;/em&gt;-nitrophenyl decanoate</td><td>Sigma</td><td>N0252</td><td>Enzymatic assays</td></tr><tr><td>&lt;em&gt;p&lt;/em&gt;-nitrophenyl palmitate</td><td>Sigma</td><td>N2752</td><td>Enzymatic assays</td></tr><tr><td>&lt;em&gt;p&lt;/em&gt;-nitrophenyl butyrate</td><td>Sigma</td><td>N9876</td><td>Enzymatic assays</td></tr><tr><td>&lt;em&gt;p&lt;/em&gt;-nitrophenyl octanoate</td><td>Sigma</td><td>21742</td><td>Enzymatic assays</td></tr><tr><td>Acetic Acid, sodium salt [1-&lt;sup&gt;14&lt;/sup&gt;C]</td><td>Perkin Elmer</td><td>NEC084</td><td>Bacterial growth</td></tr><tr><td>dimethylsulfoxide (DMSO)</td><td>JT Baker</td><td>9224-01</td><td>Enzymatic assays</td></tr><tr><td>Aluminium HPTLC silica gel 60 plates. Silica gel HPTLC plates size 20 x 20 cm, 25 sheets.</td><td>Merck</td><td>105547</td><td>TLC analysis &amp;amp; Lipid extraction</td></tr><tr><td>Spectrometer UV/VIS Lambda 35</td><td>Perkin Elmer</td><td>NA</td><td>Enzymatic assays</td></tr><tr><td>Storm 820 Phosphorimager</td><td>Molecular Dynamics</td><td>NA</td><td>Photostimulable Luminescence scanner&amp;nbsp;</td></tr><tr><td>Multipurpose Scintillation Counter</td><td>Beckman Coulter</td><td>NA</td><td>Radioactivity Quantification</td></tr><tr><td>French Pressure Cell</td><td>ThermoSpectronic</td><td>NA&amp;nbsp;</td><td>Breakage of cells</td></tr><tr><td>chromatography paper 3MM Chr</td><td>Whatman</td><td>3030917</td><td>TLC analysis</td></tr><tr><td>&lt;em&gt;Sinorhizobium meliloti&lt;/em&gt; 1021our</td><td>reference 11</td><td></td><td>studied strain</td></tr><tr><td>&lt;em&gt;Escherichia coli&lt;/em&gt; BL21 (DE3) pLysS Competent cells</td><td>Novagen</td><td>69451</td><td>protein expression strain</td></tr><tr><td>pET9a vector</td><td>Novagen</td><td>69431</td><td>protein expression vector</td></tr><tr><td>pET17b vector</td><td>Novagen</td><td>69663</td><td>protein expression vector</td></tr><tr><td>sterile polystyrene round-bottom tube (14 ml) Falcon</td><td>Becton Dickinson</td><td>352057</td><td>radiolabeling of bacterial cultures</td></tr><tr><td>polypropylene microcentrifuge tubes (1.5 ml)</td><td>Eppendorf</td><td>30125.15</td><td>Enzymatic assays</td></tr><tr><td>1,2-dipalmitoyl-&lt;em&gt;sn&lt;/em&gt;-glycerol</td><td>Sigma</td><td>D9135</td><td>lipid standard</td></tr><tr><td>L-&amp;alpha;-phosphatidylcholine, dipalmitoyl</td><td>Sigma</td><td>P6267</td><td>lipid standard</td></tr><tr><td>DL-&amp;alpha;-monopalmitin</td><td>Sigma</td><td>M1640</td><td>lipid standard</td></tr><tr><td>palmitic acid</td><td>Sigma</td><td>P0500</td><td>lipid standard</td></tr></tbody></table>

defining substrate specificities for lipase and phospholipase candidates

Mikroorganismen produzieren ein breites Spektrum von (phospho) Lipasen, die um sezerniert werden für den Organismus externe Substrate zur Verfügung zu stellen. Alternativ können andere (phospho) Lipasen physikalisch mit dem produzierenden Organismus verursacht einen Umsatz von intrinsischen Lipiden assoziiert sein können und häufig zu einem Remodeling der Zellmembranen führt. Obwohl Potential (phospho) Lipasen mit einer Anzahl von Algorithmen vorhergesagt werden, wenn das Gen / Protein-Sequenz verfügbar ist, experimentelle Nachweis der Enzymaktivitäten, Substratspezifitäten, und potentielle physiologische Funktionen ist häufig nicht erhalten werden. Dieses Manuskript beschreibt die Optimierung der Testbedingungen für Interessenten (phospho) Lipasen mit unbekannten Substratspezifität und wie diese optimierten Bedingungen bei der Suche nach dem natürlichen Substrat eines jeweiligen (phospho) Lipase zu verwenden. Mit künstlichen chromogene Substrate, wie p - Nitrophenyl - Derivate, kann dazu beitragen , eine kleinere zu erkennenenzymatische Aktivität für eine vorhergesagte (phospho) Lipase unter Standardbedingungen. Eine solche geringfügige enzymatische Aktivität angetroffen wird, können die unterschiedlichen Parameter einer Enzymassay, um einen effizienteren Hydrolyse des künstlichen Substrat zu erhalten, variiert werden. Nachdem die Bedingungen arbeitet ein Enzym gut unter denen sie festgestellt haben, sollte eine Vielzahl von potentiellen natürlichen Substrate für deren Abbau getestet werden, ein Prozess, der verschiedene chromatographische Methoden gefolgt Einsatz werden können. Die Definition von Substratspezifitäten für neue Enzyme, liefert oft Hypothesen für eine mögliche physiologische Rolle dieser Enzyme, die dann experimentell überprüft werden kann. Nach diesen Richtlinien, konnten wir eine Phospholipase C (SMc00171) , die Phosphatidylcholin, für den Umbau von Membranen in dem Bakterium Sinorhizobium meliloti auf phosphor limitierenden Bedingungen des Wachstums in einem entscheidenden Schritt phosphocholin und Diacylglycerin verschlechtert zu identifizieren. Für zwei vorhergesagten patatin-wie Phospholipasen (SMc00930 und SMc01003) des gleichen Organismus, könnten wir ihre Substratspezifität neu zu definieren und zu klären, dass SMc01003 ein Diacylglycerol Lipase ist.

Aktivität von PC-spezifische Phospholipase C SMc00171 mit Bis- p - Nitrophenylphosphat Zellfreie Extrakte von E. erhalten coli BL21 (DE3) pLysS x, die smc00171 hatte ausgedrückt, wurden auf ihre Fähigkeit untersucht bis- p - Nitrophenylphosphat Ester zu hydrolysieren, eine spektrophotometrische enzymatischen Assay unter Verwendun...

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Defining Substrate Specificities for Lipase and Phospholipase Candidates

Many predicted (phospho)lipases are poorly characterized with regard to their substrate specificities and physiological functions. Here we provide a protocol to optimize enzyme activities, search for natural substrates, and propose physiological functions for these enzymes.

Definieren von Substratspezifität für Lipase und Phospholipase-Kandidaten

Microorganisms produce a wide spectrum of (phospho)lipases that are secreted in order to make external substrates available for the organism. ...

defining-substrate-specificities-for-lipase-phospholipase

Universidad de Cartagena UDEC

Research

JoVE Journal

Biochemistry

14.9K Aufrufe.  Universidad Nacional Autónoma de México. Many predicted (phospho)lipases are poorly characterized with regard to their substrate specificities and physiological functions. Here we provide a protocol to optimize enzyme activities, search for natural substrates, and propose physiological functions for these enzymes.

Serie: Defining Substrate Specificities for Lipase and Phospholipase Candidates

A Fluorescence-based Assay of Phospholipid Scramblase Activity

Scramblases translocate phospholipids across the membrane bilayer bidirectionally in an ATP-independent manner. The first scramblase to be identified and biochemically verified was opsin, the apoprotein of the photoreceptor rhodopsin. Rhodopsin is a G protein-coupled receptor localized in rod photoreceptor disc membranes of the retina where it is responsible for the perception of light. Rhodopsin&#39;s scramblase activity does not depend on its ligand 11-cis-retinal, i.e., the apoprotein opsin is also active as a scramblase. Although constitutive and regulated phospholipid scrambling play an important role in cell physiology, only a few phospholipid scramblases have been identified so far besides opsin. Here we describe a fluorescence-based assay of opsin&#39;s scramblase activity. Opsin is reconstituted into large unilamellar liposomes composed of phosphatidylcholine, phosphatidylglycerol and a trace quantity of fluorescent NBD-labeled PC (1-palmitoyl-2-{6-[7-nitro-2-1,3-benzoxadiazole-4-yl)amino]hexanoyl}-sn-glycero-3-phosphocholine). Scramblase activity is determined by measuring the extent to which NBD-PC molecules located in the inner leaflet of the vesicle are able to access the outer leaflet where their fluorescence is chemically eliminated by a reducing agent that cannot cross the membrane. The methods we describe have general applicability and can be used to identify and characterize scramblase activities of other membrane proteins.

We describe a fluorescence-based assay to measure phospholipid scrambling in large unilamellar liposomes reconstituted with opsin.

Ein Fluoreszenz-basierte Assay von Phospholipid Scramblase Activity

Scramblases translocate phospholipids across the membrane bilayer bidirectionally in an ATP-independent manner. The first scramblase to be identified and ...

Forschung

Biochemie

Unraveling Entropic Rate Acceleration Induced by Solvent Dynamics in Membrane Enzymes

Enzyme catalysis evolved in an aqueous environment. The influence of solvent dynamics on catalysis is, however, currently poorly understood and usually neglected. The study of water dynamics in enzymes and the associated thermodynamical consequences is highly complex and has involved computer simulations, nuclear magnetic resonance (NMR) experiments, and calorimetry. Water tunnels that connect the active site with the surrounding solvent are key to solvent displacement and dynamics. The protocol herein allows for the engineering of these motifs for water transport, which affects specificity, activity and thermodynamics. By providing a biophysical framework founded on theory and experiments, the method presented herein can be used by researchers without previous expertise in computer modeling or biophysical chemistry. The method will advance our understanding of enzyme catalysis on the molecular level by measuring the enthalpic and entropic changes associated with catalysis by enzyme variants with obstructed water tunnels. The protocol can be used for the study of membrane-bound enzymes and other complex systems. This will enhance our understanding of the importance of solvent reorganization in catalysis as well as provide new catalytic strategies in protein design and engineering.

Channels for the transportation of water molecules in enzymes influence active site solvation and catalysis. Herein we present a protocol for the engineering of these additional catalytic motifs based on in silico computer modeling and experiments. This will enhance our understanding of the influence of solvent dynamics on enzyme catalysis.

Entwirren Entropic Reaktionsbeschleunigung durch Solvent Dynamics in Membranenzyme induziert

Enzyme catalysis evolved in an aqueous environment. The influence of solvent dynamics on catalysis is, however, currently poorly understood and usually ...

Chemie

The Determination of Protease Specificity in Mouse Tissue Extracts by MALDI-TOF Mass Spectrometry: Manipulating PH to Cause Specificity Changes

Proteases have several biological functions, including protein activation/inactivation and food digestion. Identifying protease specificity is important for revealing protease function. The method proposed in this study determines protease specificity by measuring the molecular weight of unique substrates using Matrix-assisted Laser Desorption/Ionization Time-of-Flight (MALDI-TOF) mass spectrometry. The substrates contain iminobiotin, while the cleaved site consists of amino acids, and the spacer consists of polyethylene glycol. The cleaved substrate will generate a unique molecular weight using a cleaved amino acid. One of the merits of this method is that it may be carried out in one pot using crude samples, and it is also suitable for assessing multiple samples. In this article, we describe a simple experimental method optimized with samples extracted from mouse lung tissue, including tissue extraction, placement of digestive substrates into samples, purification of digestive substrates under different pH conditions, and measurement of the substrates&#39; molecular weight using MALDI-TOF mass spectrometry. In summary, this technique allows for the identification of protease specificity in crude samples derived from tissue extracts using MALDI-TOF mass spectrometry, which may easily be scaled up for multiple sample processing.

Here, we present a protocol to determine protease specificity in crude mouse tissue extracts using MALDI-TOF spectrometry.

Die Bestimmung der Protease Spezifität im Maus-Gewebe extrahiert durch MALDI-TOF-Massenspektrometrie: PH um Spezifität Änderungen führen zu manipulieren

Proteases have several biological functions, including protein activation/inactivation and food digestion. Identifying protease specificity is important ...

Use of Microscale Thermophoresis to Measure Protein-Lipid Interactions

The ability to determine the binding affinity of lipids to proteins is an essential part of understanding protein-lipid interactions in membrane trafficking, signal transduction and cytoskeletal remodeling. Classic tools for measuring such interactions include surface plasmon resonance (SPR) and isothermal titration calorimetry (ITC). While powerful tools, these approaches have setbacks. ITC requires large amounts of purified protein as well as lipids, which can be costly and difficult to produce. Furthermore, ITC as well as SPR are very time consuming, which could add significantly to the cost of performing these experiments. One way to bypass these restrictions is to use the relatively new technique of microscale thermophoresis (MST). MST is fast and cost effective using small amounts of sample to obtain a saturation curve for a given binding event. There currently are two types of MST systems available. One type of MST requires labeling with a fluorophore in the blue or red spectrum. The second system relies on the intrinsic fluorescence of aromatic amino acids in the UV range. Both systems detect the movement of molecules in response to localized induction of heat from an infrared laser. Each approach has its advantages and disadvantages. Label-free MST can use untagged native proteins; however, many analytes, including pharmaceuticals, fluoresce in the UV range, which can interfere with determination of accurate KD values. In comparison, labeled MST allows for a greater diversity of measurable pairwise interactions utilizing fluorescently labeled probes attached to ligands with measurable absorbances in the visible range as opposed to UV, limiting the potential for interfering signals from analytes.

Microscale thermophoresis obtains binding constants quickly at low material cost. Either labeled or label free microscale thermophoresis is commercially available; however, label free thermophoresis is not capable of the diversity of interaction measurements that can be performed using fluorescent labels. We provide a protocol for labeled thermophoresis measurements.

Einsatz der mikroskaligen Thermophorese zur Messung von Protein-Lipid-Wechselwirkungen

The ability to determine the binding affinity of lipids to proteins is an essential part of understanding protein-lipid interactions in membrane ...

Click-Chemistry Based Fluorometric Assay for Apolipoprotein N-acyltransferase from Enzyme Characterization to High-Throughput Screening

Lipoproteins from proteobacteria are posttranslationally modified by fatty acids derived from membrane phospholipids by the action of three integral membrane enzymes, resulting in triacylated proteins. The first step in the lipoprotein modification pathway involves the transfer of a diacylglyceryl group from phosphatidylglycerol onto the prolipoprotein, resulting in diacylglyceryl prolipoprotein. In the second step, the signal peptide of prolipoprotein is cleaved, forming an apolipoprotein, which in turn is modified by a third fatty acid derived from a phospholipid. This last step is catalyzed by apolipoprotein N-acyltransferase (Lnt). The lipoprotein modification pathway is essential in most &#947;-proteobacteria, making it a potential target for the development of novel antibacterial agents. Described here is a sensitive assay for Lnt that is compatible with high-throughput screening of small inhibitory molecules. The enzyme and substrates are membrane-embedded molecules; therefore, the development of an in vitro test is not straightforward. This includes the purification of the active enzyme in the presence of detergent, the availability of alkyne-phospholipids and diacylglyceryl peptide substrates, and the reaction conditions in mixed micelles. Furthermore, in order to use the activity test in a high-throughput screening (HTS) setup, direct readout of the reaction product is preferred over coupled enzymatic reactions. In this fluorometric enzyme assay, the alkyne-triacylated peptide product is rendered fluorescent through a click-chemistry reaction and detected in a multiwell plate format. This method is applicable to other acyltransferases that use fatty acid-containing substrates, including phospholipids and acyl-CoA.

Presented here is a sensitive fluorescence assay to monitor apolipoprotein N-acyltransferase activity using diacylglyceryl peptide and alkyne-phospholipids as substrates with click-chemistry.

Klickchemischer fluorometrischer Assay für Apolipoprotein-N-Acyltransferase von der Enzymcharakterisierung bis zum Hochdurchsatz-Screening

Lipoproteins from proteobacteria are posttranslationally modified by fatty acids derived from membrane phospholipids by the action of three integral ...

Fluorescence-Based Measurements of Phosphatidylserine/Phosphatidylinositol 4-Phosphate Exchange Between Membranes

Several members of the evolutionarily conserved oxysterol-binding protein (OSBP)-related proteins(ORP)/OSBP homologs (Osh) family have recently been found to represent a novel lipid transfer protein (LTP) group in yeast and human cells. They transfer phosphatidylserine (PS) from the endoplasmic reticulum (ER) to the plasma membrane (PM) via PS/phosphatidylinositol 4-phosphate (PI(4)P) exchange cycles. This finding allows a better understanding of how PS, which is critical for signaling processes, is distributed throughout the cell and the investigation of the link between this process and phosphoinositide (PIP) metabolism. The development of new fluorescence-based protocols has been instrumental in the discovery and characterization of this new cellular mechanism in vitro at the molecular level. This paper describes the production and the use of two fluorescently labelled lipid sensors, NBD-C2Lact and NBD-PHFAPP, to measure the ability of a protein to extract PS or PI(4)P and to transfer these lipids between artificial membranes. First, the protocol describes how to produce, label, and obtain high-purity samples of these two constructs. Secondly, this paper explains how to use these sensors with a fluorescence microplate reader to determine whether a protein can extract PS or PI(4)P from liposomes, using Osh6p as a case study. Finally, this protocol shows how to accurately measure the kinetics of PS/PI(4)P exchange between liposomes of defined lipid composition and to determine lipid transfer rates by fluorescence resonance energy transfer (FRET) using a standard fluorometer.

Here, we describe protocols using fluorescent lipid sensors and liposomes to determine whether a protein extracts and transports phosphatidylserine or phosphatidylinositol 4-phosphate in vitro.

Fluoreszenzbasierte Messungen des Phosphatidylserin/Phosphatidylinositol-4-Phosphat-Austauschs zwischen Membranen

Several members of the evolutionarily conserved oxysterol-binding protein (OSBP)-related proteins(ORP)/OSBP homologs (Osh) family have recently been found ...

Analysis of Lipid Signaling in Drosophila Photoreceptors using Mass Spectrometry

The activation of phospholipase C&#946; (PLC&#946;) is an essential step during sensory transduction in Drosophila photoreceptors. PLC&#946; activity results in the hydrolysis of the membrane lipid phosphatidylinositol 4,5 bisphosphate [PI(4,5)P2] leading ultimately to the activation of transient receptor potential (TRP) and TRP like (TRPL) channels. The activity of PLC&#946; also leads subsequently to the generation of many lipid species several of which have been proposed to play a role in TRP and TRPL activation. In addition, several classes of lipids have been proposed to play key roles in organizing the cell biology of photoreceptors to optimize signaling reactions for optimal sensory transduction. Historically, these discoveries have been driven by the ability to isolate Drosophila mutants for enzymes that control the levels of specific lipids and perform analysis of photoreceptor physiology in these mutants. More recently, powerful mass spectrometry methods for isolation and quantitative analysis of lipids with high sensitivity and specificity have been developed. These are particularly suited for use in Drosophila where lipid analysis is now possible from photoreceptors without the need for radionuclide labeling. In this article, the conceptual and practical considerations in the use of lipid mass spectrometry for the robust, sensitive, and accurate quantitative assessment of various signaling lipids in Drosophila photoreceptors are covered. Along with existing methods in molecular genetics and physiological analysis such lipid is likely to enhance the power of photoreceptors as a model system for discoveries in biology.

Analysis of Lipid Signaling in Drosophila Photoreceptors using Mass Spectrometry

The manuscript presents versatile, robust, and sensitive mass spectrometry protocols to identify and quantify several classes of lipids from Drosophila photoreceptors.

The activation of phospholipase C&#946; (PLC&#946;) is an essential step during sensory transduction in Drosophila photoreceptors. PLC&#946; activity ...

PIP-on-a-chip: A Label-free Study of Protein-phosphoinositide Interactions

Numerous cellular proteins interact with membrane surfaces to affect essential cellular processes. These interactions can be directed towards a specific lipid component within a membrane, as in the case of phosphoinositides (PIPs), to ensure specific subcellular localization and/or activation. PIPs and cellular PIP-binding domains have been studied extensively to better understand their role in cellular physiology. We applied a pH modulation assay on supported lipid bilayers (SLBs) as a tool to study protein-PIP interactions. In these studies, pH sensitive ortho-Sulforhodamine B conjugated phosphatidylethanolamine is used to detect protein-PIP interactions. Upon binding of a protein to a PIP-containing membrane surface, the interfacial potential is modulated (i.e. change in local pH), shifting the protonation state of the probe. A case study of the successful usage of the pH modulation assay is presented by using phospholipase C delta1 Pleckstrin Homology (PLC-&#948;1 PH) domain and phosphatidylinositol 4,5-bisphosphate (PI(4,5)P2) interaction as an example. The apparent dissociation constant (Kd,app) for this interaction was 0.39 &#177; 0.05 &#181;M, similar to Kd,app values obtained by others. As previously observed, the PLC-&#948;1 PH domain is PI(4,5)P2 specific, shows weaker binding towards phosphatidylinositol 4-phosphate, and no binding to pure phosphatidylcholine SLBs. The PIP-on-a-chip assay is advantageous over traditional PIP-binding assays, including but not limited to low sample volume and no ligand/receptor labeling requirements, the ability to test high- and low-affinity membrane interactions with both small and large molecules, and improved signal to noise ratio. Accordingly, the usage of the PIP-on-a-chip approach will facilitate the elucidation of mechanisms of a wide range of membrane interactions. Furthermore, this method could potentially be used in identifying therapeutics that modulate protein&#39;s capacity to interact with membranes.

Here we present a supported lipid bilayer in the context of a microfluidic platform to study protein-phosphoinositide interactions using a label-free method based on pH modulation.

PIP-on-a-Chip: Eine Label-freie Studie von Protein-Phosphoinositid-Wechselwirkungen

Numerous cellular proteins interact with membrane surfaces to affect essential cellular processes. These interactions can be directed towards a specific ...

Biotechnik

Concanavalin A-Based Sedimentation Assay to Measure Substrate Binding of Glucan Phosphatases

Glucan phosphatases belong to the larger family of dual specificity phosphatases (DSP) that dephosphorylate glucan substrates, such as glycogen in animals and starch in plants. The crystal structures of glucan phosphatase with model glucan substrates reveal distinct glucan-binding interfaces made of DSP and carbohydrate-binding domains. However, quantitative measurements of glucan-glucan phosphatase interactions with physiologically relevant substrates are fundamental to the biological understanding of the glucan phosphatase family of enzymes and the regulation of energy metabolism. This manuscript reports a Concanavalin A (ConA)-based in vitro sedimentation assay designed to detect the substrate binding affinity of glucan phosphatases against different glucan substrates.&#160;As a proof of concept, the dissociation constant (KD) of glucan phosphatase Arabidopsis thaliana Starch Excess4 (SEX4) and amylopectin was determined. The characterization of SEX4 mutants and other members of the glucan phosphatase family of enzymes further demonstrates the utility of this assay to assess the differential binding of protein- carbohydrate interactions. These data demonstrate the suitability of this assay to characterize a wide range of starch and glycogen interacting proteins.

This method describes a lectin-based in vitro sedimentation assay to quantify the binding affinity of glucan phosphatase and amylopectin. This co-sedimentation assay is reliable for measuring glucan phosphatase substrate binding and can be applied to various solubilized glucan substrates.

Concanavalin A-basierter Sedimentationsassay zur Messung der Substratbindung von Glucanphosphatasen

Glucan phosphatases belong to the larger family of dual specificity phosphatases (DSP) that dephosphorylate glucan substrates, such as glycogen in animals ...

Construction of Model Lipid Membranes Incorporating G-protein Coupled Receptors (GPCRs)

Robust in vitro investigations of the structure and function of integral membrane proteins has been a challenge due to the complexities of the plasma membrane and the numerous factors that influence protein behavior in live cells. Giant unilamellar vesicles (GUVs) are a biomimetic and highly tunable in vitro model system for investigating protein-membrane interactions and probing protein behavior in a precise, stimulus-dependent manner. In this protocol, we present an inexpensive and effective method for fabricating GUVs with the human serotonin 1A receptor (5-HT1AR) stably integrated in the membrane. We fabricate GUVs using a modified hydrogel swelling method; by depositing a lipid film on top of a mixture of agarose and 5-HT1AR and then hydrating the entire system, vesicles can be formed with properly oriented and functional 5-HT1AR incorporated into the membrane. These GUVs can then be used to examine protein-membrane interactions and localization behavior via microscopy. Ultimately, this protocol can advance our understanding of the functionality of integral membrane proteins, providing profound physiological insight.

Construction of Model Lipid Membranes Incorporating G-protein Coupled Receptors GPCRs

This protocol utilizes agarose swelling as a powerful and generalizable technique for incorporating integral membrane proteins (IMPs) into giant unilamellar lipid vesicles (GUVs), as described here for the reconstitution of the human 1A serotonin receptor protein (5-HT1AR), one of the classes of pharmacologically important G protein-coupled receptors.

Konstruktion von Modelllipidmembranen mit G-Protein-gekoppelten Rezeptoren (GPCRs)

Robust in vitro investigations of the structure and function of integral membrane proteins has been a challenge due to the complexities of the plasma ...

Definieren von Substratspezifität für Lipase und Phospholipase-Kandidaten

Zusammenfassung

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Die Bestimmung der Protease Spezifität im Maus-Gewebe extrahiert durch MALDI-TOF-Massenspektrometrie: PH um Spezifität Änderungen führen zu manipulieren

Einsatz der mikroskaligen Thermophorese zur Messung von Protein-Lipid-Wechselwirkungen

Klickchemischer fluorometrischer Assay für Apolipoprotein-N-Acyltransferase von der Enzymcharakterisierung bis zum Hochdurchsatz-Screening

Fluoreszenzbasierte Messungen des Phosphatidylserin/Phosphatidylinositol-4-Phosphat-Austauschs zwischen Membranen

Analysis of Lipid Signaling in Drosophila Photoreceptors using Mass Spectrometry

PIP-on-a-Chip: Eine Label-freie Studie von Protein-Phosphoinositid-Wechselwirkungen

Concanavalin A-basierter Sedimentationsassay zur Messung der Substratbindung von Glucanphosphatasen

Konstruktion von Modelllipidmembranen mit G-Protein-gekoppelten Rezeptoren (GPCRs)