이 작품은 (Investigación 엉 Fronteras 드 라 Ciencia에서 Investigación Científica BASICA에서 82614, 153998, 253549 및 178359뿐만 아니라 118) 및 Dirección 일반 드에서 Consejo 나시 오날 드를 CIENCIAS Y TECNOLOGIA - 멕시코 (CONACYT 멕시코)에서 보조금에 의해 지원되었다 (DGAPA - UNAM, PAPIIT IN202616, IN203612) Asuntos 드 개인 Académico-대학교 나시 오날 자치시 드 멕시코.

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저자는 공개 아무것도 없어.

지난 20 년 동안, 많은 생물의 게놈은 서열화되었다 및 게놈 시퀀스 데이터의 재산이 생성되었지만, 기능 해석은 뒤쳐 따라서 게놈 기능에 대한 우리의 이해를 방해한다. 게놈에서 유전자의 기능은 종종 알려진 기능 또는 보존 모티브의 발생 유전자의 유사성을 기준으로 할당됩니다. 그러나, 특정 유전자의 정확한 기능은 대개 알려져 있지 않다. 특히, 효소 구조 유전자를 쉽게 인해 대부분의 효소...

이러한 트리 아실 글리세롤과 (글리세) 인지질 등의 글리세롤 기반의 지질이 중요한 구성하고 아마 가장 잘 알려진 지질 클래스 1. 트리 아실 글리세롤 (태그) 지방 또는 일반적으로 저장 지질의 기능 오일, 따라서 에너지 및 탄소 소스로합니다. 태그는 자주 외부 태그를 소화하고 탄소 소스로이를 사용할 수 있도록 생산 미생물에 의해 분비되는 리파제에 의해 저하 될 수 있습니다. 또한, 리파제 널리 그들의 중요한 생명 공학 응용 프로그램을 2 년 동안 연구되어왔다. 때문에 자신의 양친 매성 자연과 가까운 원통 모양, (글리세) 인지질 전시 막 형성 특성과 일반적으로 이중 층 막 3의 주요 지질 구성 요소를 구성합니다. 이러한 박테리아, 대장균, 세 개의 주요 그룹 헤드 변형, 포스파티딜 글리세롤 (PG), 카디오 리핀 (CL) 및 phosphatid 간단한 미생물에서하나는 그들 각자가 서로 다른 분자 종 (4)를 다수로 상기 SN의 다른 지방산 아실 사슬의 상당수 -1 SN -2 위치주는 증가로 치환 될 수 있음을 알아야하지만 ylethanolamine (PE)가 발생하는 . 다른 박테리아는 다른 인지질 외에 또는 대신이있을 수 있습니다. 예를 들어, Sinorhizobium의 meliloti은, 콩과 식물 알팔파 (Medicago 사티)와 질소 고정 뿌리혹 공생 관계를 형성 할 수있는 토양 박테리아, 제 양쪽이 온성 인지질, 포스파티딜콜린 (PC)를 PE 이외에 포함 오. 또한, 지질이 포함되지 인 또는 글리세롤은 세포막의 양친과 형태의 일부가 될 수 있습니다. 예를 들어, 인 - 제한 성장 조건에 따라, S.의 meliloti, (글리세) 인지질은 크게, 즉, sulfolipids, 오르니 틴 지질 및 diacylglyceryl의 trimethylho 인을 포함하지 않는 막 지질로 대체moserine (DGTS) 6. 박테리아, DGTS은 두 단계 경로 7에서 디아 실 글리세롤 (DAG)로 형성되지만 DAG 생성하기위한 소스는 명확하지 않았다된다. 펄스 - 체이스 실험은 PC가 인 제한 조건에서 형성된다 및 DAG 및 포스 포 콜린에 PC를 변환 할 수있는 DGTS 8 전구체하고 우리는 포스 포 리파제 C (PLCP, SMc00171)을 식별 할 수있는이 원고에 기술 된 방법을 사용하여 수 있다는 제안 8. 별도의 연구에서 우리는 발견이 아 닐 -CoA 합성 효소 (FADD) S.의 결핍 돌연변이 성장 9 정지상 들어갈 때 meliloti 또는 대장균 유리 지방산 축적. 이들 지방산은 세포막의 지질로부터 유도 될 듯하지만, 유리 지방산 또는이를 유리화하는 효소 (들)에 대한 정확한 소스는 공지되지 않았다. 다시 말하지만, 전략을 사용하는 것은이 원고에 설명 된 두 파타 틴과 같은 10 (포스) 리파제 (S.에서 유리 지방산의 형성에 기여하고 SMc00930 SMc01003) meliloti (11)는 예측했다. 놀랍게도 SMc01003는 글리세롤 및 유리 지방산 및 모노 아실 글리세롤 마지막 11로 변환 기판으로서 DAG를 이용했다. 따라서, SMc01003는 DAG 리파제 (DGLA)입니다. 알고리즘의 숫자가 전위 (포스)를 예측하는 존재하지만 12, 13는, 리파제 자신의 정확한 기능과 생리 학적 역할은 일반적으로 알려져 있지 않다. 여기에서 우리는 복제, 프로토콜 개요 및 예측 또는 전위 (포스) 리파제를 과발현. 이 원고 효소 분석법 개발 인공 발색 기질을 사용하여 과발현 (포스) 리파제에 최적화 할 수있는 방법을 설명한다. 우리는이 연구 결과는 미생물 생리학에 대한 우리의 이해를 풍부하게하는 방법을 최적화 효소 분석과 실제 (포스) 리파제 기판이 발생할 수있는 방법과 예제를 제공합니다.

<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

미생물 유기체의 외부 기판을 사용할 수 있도록하기 위해 분비된다 (포스) 리파제의 넓은 스펙트럼을 생산하고 있습니다. 또한, 다른 (포스) 리파제는 물리적으로 생산 유기체가 고유 지질의 매출을 일으키는 원인이 자주 세포막의 리모델링에 상승을 제공과 연관 될 수있다. 전위 (포스)은 리파제 유전자 / 단백질 서열 사용할 알고리즘의 번호와 예측 될 수 있지만, 효소 활성, 기질 특이성, 잠재적 생리 기능의 실험적 증거는 종종 수득되지 않았다. 이 원고는 알 수없는 기질 특이성하는 방법과 각각의 (포스) 리파제의 자연 기판에 대한 검색이 최적의 조건을 사용하는 방법과 미래 (포스) 리파제에 대한 분석 조건의 최적화에 대해 설명합니다. 미성년자를 감지하는 데 도움이, 같은 페이지의 -nitrophenyl 유도체 인공 발색 기판을, 수 사용표준 조건에서 예측 (포스) 리파제에 대한 효소 활성. 이러한 보조 효소 활성을 발생하는 데, 효소 분석의 고유 파라미터는 인공 기판의보다 효율적인 가수 분해를 얻기 위해 변화 될 수있다. 효소가 잘 작동되는 조건을 판정 한 후, 자연 전위 다양한 기판은 그들의 열화 구별 크로마토 그래피 방법을 이용 하였다 될 수있는 프로세스를 분석한다. 새로운 효소 기질 특이성의 정의는 종종 후 실험적으로 테스트 될 수있는 이러한 효소의 전위 생리적 역할에 대한 가설을 제공한다. 이 가이드 라인에 따라, 우리는 성장의 인 제한 조건에 따라 세균 Sinorhizobium의 meliloti에서 세포막의 리모델링을위한 중요한 단계에서, 포스 포 콜린과 디아 실 글리세롤하는 포스파티딜콜린을 저하 포스 포 리파제 C (SMc00171)을 식별 할 수 있었다. 두 patatin- 예측을 위해같은 유기체의 포스 포 (SMc00930 및 SMc01003)처럼, 우리는 그들의 기질 특이성을 재정의하고 SMc01003은 디아 실 글리세롤 리파제 있음을 명확히 할 수있다.

비스 P는 -nitrophenyl 인산와 PC 고유의 포스 C SMc00171의 활동 E. 얻은 무 세포 추출물 대장균 BL21 (DE3)는 smc00171가 형성된 P는 -NP을 측정하는 분광 효소 분석을 이용하여, 비스 페이지 -nitrophenyl 인산 에스테르를 가수 분해하는 ?...

Watch this Scientific Journal Video about Defining Substrate Specificities for Lipase and Phospholipase Candidates at JoVE.com

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.

리파제 및 포스 후보자에 대한 기질 특이성을 정의

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

Research

JoVE Journal

Biochemistry

14.9K 조회수.  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.

비디오: 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.

인지질 Scramblase 활동의 형광 기반의 분석

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

연구

생화학

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.

막 효소 용매 역학에 의해 유도 탈피 엔트 속도 가속

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

화학

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.

마우스 조직에서 효소 특이성 결정 TOF MALDI 질량 분석에 의해 추출: PH 특이성 변경를 조작

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.

단백질-지질 상호작용을 측정하기 위한 마이크로스케일 열영동 사용

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.

효소 특성화에서 고처리량 스크리닝까지 아포지단백질 N-아실트랜스퍼라제에 대한 클릭 화학 기반 형광 분석

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.

형광 기반 측정 인스파디딜세린/포스파디딜리노시톨 4-인산염 교환 막 간

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 : 단백질 - 포스 포이 노시 티드 상호 작용에 대한 라벨없는 연구

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

생체공학

In vitro Digestion of Emulsions in a Single Droplet via Multi Subphase Exchange of Simulated Gastrointestinal Fluids

Emulsions are currently being used to encapsulate and deliver nutrients and drugs to tackle different gastrointestinal conditions such as obesity, nutrient fortification, food allergies, and digestive diseases. The ability of an emulsion to provide the desired functionality, namely, reaching a specific site within the gastrointestinal tract, inhibiting/retarding lipolysis, or facilitating digestibility, ultimately depends on its susceptibility to enzymatic degradation in the gastrointestinal tract. In oil-in-water emulsions, lipid droplets are surrounded by interfacial layers, where the emulsifiers stabilize the emulsion and protect the encapsulated compound. Achieving a tailored digestibility of emulsions depends on their initial composition but also requires monitoring the evolution of those interfacial layers as they are subjected to different phases of gastrointestinal digestion. A pendant drop surface film balance implemented with a multi-subphase exchange allows for simulating the in vitro digestion of emulsions in a single aqueous droplet immersed in oil by applying a customized static digestion model. The transit through the gastrointestinal tract is mimicked by the subphase exchange of the original droplet bulk solution with artificial media, mimicking the physiological conditions of each compartment/step of the gastrointestinal tract. The dynamic evolution of the interfacial tension is recorded in situ throughout the whole simulated gastrointestinal digestion. The mechanical properties of digested interfaces, such as interfacial dilatational elasticity and viscosity, are measured after each digestion phase (oral, gastric, small intestine). The composition of each digestive media can be tuned to account for the particularities of the digestive conditions, including gastrointestinal pathologies and infant digestive media. The specific interfacial mechanisms affecting proteolysis and lipolysis are identified, providing tools to modulate digestion by the interfacial engineering of emulsions. The obtained results can be manipulated for designing novel food matrices with tailored functionalities such as low allergenicity, controlled energy intake, and decreased digestibility.

In vitro Digestion of Emulsions in a Single Droplet via Multi Subphase Exchange of Simulated Gastrointestinal Fluids

A pendant drop surface film balance implemented with a multi-subphase exchange, nicknamed the OCTOPUS, allows for mimicking digestive conditions by the sequential subphase exchange of the original bulk solution with simulated gastrointestinal fluids. The simulated in vitro digestion is monitored by recording in situ the interfacial tension of the digested interfacial layer.

시험관 내 시뮬레이션된 위장관액의 다중 하위 단계 교환 을 통한 단일 액적의 에멀젼 소화

Emulsions are currently being used to encapsulate and deliver nutrients and drugs to tackle different gastrointestinal conditions such as obesity, ...

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.

G 단백질 결합 수용체 (GPCRs)를 통합 한 모델 지질 막의 건설

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

리파제 및 포스 후보자에 대한 기질 특이성을 정의

요약

더 많은 비디오 탐색

이 비디오의 챕터

인지질 Scramblase 활동의 형광 기반의 분석

막 효소 용매 역학에 의해 유도 탈피 엔트 속도 가속

마우스 조직에서 효소 특이성 결정 TOF MALDI 질량 분석에 의해 추출: PH 특이성 변경를 조작

단백질-지질 상호작용을 측정하기 위한 마이크로스케일 열영동 사용

효소 특성화에서 고처리량 스크리닝까지 아포지단백질 N-아실트랜스퍼라제에 대한 클릭 화학 기반 형광 분석

형광 기반 측정 인스파디딜세린/포스파디딜리노시톨 4-인산염 교환 막 간

Analysis of Lipid Signaling in Drosophila Photoreceptors using Mass Spectrometry

PIP-on-a-chip : 단백질 - 포스 포이 노시 티드 상호 작용에 대한 라벨없는 연구

시험관 내 시뮬레이션된 위장관액의 다중 하위 단계 교환 을 통한 단일 액적의 에멀젼 소화

G 단백질 결합 수용체 (GPCRs)를 통합 한 모델 지질 막의 건설