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.

<ol>
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Physiol. 37 (8), 911-917 (1959).</li><li>Molecular Dynamics. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Phosphorimager SI User&#180;s Guide. , (1994).</li><li>Dixon, M., Webb, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Enzymes: Third Edition. , (1979).</li><li>Rudolph, A. E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Expression,+characterization,+and+mutagenesis+of+the+Yersinia+pestis+murine+toxin,+a+phospholipase+D+superfamily+member.">Expression, characterization, and mutagenesis of the Yersinia pestis murine toxin, a phospholipase D superfamily member.</a> J. Biol. Chem. 274 (17), 11824-11831 (1999).</li><li>Kato, S., Yoshimura, T., Hemmi, H., Moriyama, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Biochemical+analysis+of+a+novel+lipolytic+enzyme+YvdO+from+Bacillus+subtilis.">Biochemical analysis of a novel lipolytic enzyme YvdO from Bacillus subtilis.</a> Biosci. Biotechnol. Biochem. 74 (4), 701-706 (2010).</li><li>Peppelenbosch, M. 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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 border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Chloroform</td>
			<td>JT Baker</td>
			<td>9180-03</td>
			<td>TLC analysis &#38; Lipid extraction</td>
		</tr>
		<tr>
			<td>Methanol</td>
			<td>JT Baker</td>
			<td>9070-03</td>
			<td>TLC analysis &#38; Lipid extraction</td>
		</tr>
		<tr>
			<td>Acetic Acid</td>
			<td>JT Baker</td>
			<td>9507-05</td>
			<td>TLC analysis &#38; Lipid extraction</td>
		</tr>
		<tr>
			<td>Hexanes</td>
			<td>JT Baker</td>
			<td>9309-02</td>
			<td>TLC analysis &#38; 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>&#160;ANY</td>
			<td>&#160;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, 10g tryptone + 5g yeast extract + 10g 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, 8g tryptone + 3g yeast extract + 66mg CaCl2 2 H20 per liter of bidistilled water</td>
		</tr>
		<tr>
			<td>CaCl2 2 H2O</td>
			<td>Baker</td>
			<td>1332-01</td>
			<td>Enzymatic assays</td>
		</tr>
		<tr>
			<td>isopropyl-&#946;-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>MnCl2</td>
			<td>Sigma</td>
			<td>221279</td>
			<td>Enzymatic assays</td>
		</tr>
		<tr>
			<td>Phospholipase A2 snake venom</td>
			<td>Sigma</td>
			<td>P0790</td>
			<td>Enzymatic assays</td>
		</tr>
		<tr>
			<td>Phospholipase C Clostridium perfingrens</td>
			<td>Sigma</td>
			<td>P7633</td>
			<td>Enzymatic assays</td>
		</tr>
		<tr>
			<td>Bis-p-nitrophenyl phosphate</td>
			<td>Sigma</td>
			<td>07422AH</td>
			<td>Enzymatic assays</td>
		</tr>
		<tr>
			<td>p-nitrophenyl stearate</td>
			<td>Sigma</td>
			<td>N3627</td>
			<td>Enzymatic assays</td>
		</tr>
		<tr>
			<td>p-nitrophenyl dodecanoate</td>
			<td>Sigma</td>
			<td>61716</td>
			<td>Enzymatic assays</td>
		</tr>
		<tr>
			<td>p-nitrophenyl decanoate</td>
			<td>Sigma</td>
			<td>N0252</td>
			<td>Enzymatic assays</td>
		</tr>
		<tr>
			<td>p-nitrophenyl palmitate</td>
			<td>Sigma</td>
			<td>N2752</td>
			<td>Enzymatic assays</td>
		</tr>
		<tr>
			<td>p-nitrophenyl butyrate</td>
			<td>Sigma</td>
			<td>N9876</td>
			<td>Enzymatic assays</td>
		</tr>
		<tr>
			<td>p-nitrophenyl octanoate</td>
			<td>Sigma</td>
			<td>21742</td>
			<td>Enzymatic assays</td>
		</tr>
		<tr>
			<td>Acetic Acid, sodium salt [1-14C]</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 &#38; 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&#160;</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&#160;</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>Sinorhizobium meliloti 1021our</td>
			<td>reference 34</td>
			<td></td>
			<td>studied strain</td>
		</tr>
		<tr>
			<td>Escherichia coli 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-sn-glycerol</td>
			<td>Sigma</td>
			<td>D9135</td>
			<td>lipid standard</td>
		</tr>
		<tr>
			<td>L-&#945;-phosphatidylcholine, dipalmitoyl</td>
			<td>Sigma</td>
			<td>P6267</td>
			<td>lipid standard</td>
		</tr>
		<tr>
			<td>DL-&#945;-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

Universidad de Cartagena UDEC

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

High-throughput Screening of Carbohydrate-degrading Enzymes Using Novel Insoluble Chromogenic Substrate Assay Kits

Carbohydrates active enzymes (CAZymes) have multiple roles in vivo and are widely used for industrial processing in the biofuel, textile, detergent, paper and food industries. A deeper understanding of CAZymes is important from both fundamental biology and industrial standpoints. Vast numbers of CAZymes exist in nature (especially in microorganisms) and hundreds of thousands have been cataloged and described in the carbohydrate active enzyme database (CAZy). However, the rate of discovery of putative enzymes has outstripped our ability to biochemically characterize their activities. One reason for this is that advances in genome and transcriptome sequencing, together with associated bioinformatics tools allow for rapid identification of candidate CAZymes, but technology for determining an enzyme's biochemical characteristics has advanced more slowly. To address this technology gap, a novel high-throughput assay kit based on insoluble chromogenic substrates is described here. Two distinct substrate types were produced: Chromogenic Polymer Hydrogel (CPH) substrates (made from purified polysaccharides and proteins) and Insoluble Chromogenic Biomass (ICB) substrates (made from complex biomass materials). Both CPH and ICB substrates are provided in a 96-well high-throughput assay system. The CPH substrates can be made in four different colors, enabling them to be mixed together and thus increasing assay throughput. The protocol describes a 96-well plate assay and illustrates how this assay can be used for screening the activities of enzymes, enzyme cocktails, and broths.

A high-throughput assay for enzyme screening is described. This multiplexed ready-to-use assay kit comprises of pre-chosen Chromogenic Polymer Hydrogel (CPH) substrates and complex Insoluble Chromogenic Biomass (ICB) substrates. Target enzymes are polysaccharide degrading endo-enzymes and proteases.

소설 불용성 발색 기질 분석 키트를 사용하여 탄수화물 분해 효소의 높은 처리량 검열

Carbohydrates active enzymes (CAZymes) have multiple roles in vivo and are widely used for industrial processing in the biofuel, textile, detergent, paper ...

연구

생화학

Biochemical and Structural Characterization of the Carbohydrate Transport Substrate-binding-protein SP0092

Development of new antimicrobials and vaccines for Streptococcus pneumoniae (pneumococcus) are necessary to halt the rapid rise in multiple resistant strains. Carbohydrate substrate binding proteins (SBPs) represent viable targets for the development of protein-based vaccines and new antimicrobials because of their extracellular localization and the centrality of carbohydrate import for pneumococcal metabolism, respectively. Described here is a rationalized integrated protocol to carry out a comprehensive characterization of SP0092, which can be extended to other carbohydrate SBPs from the pneumococcus and other bacteria. This procedure can aid the structure-based design of inhibitors for this class of proteins. Presented in the first part of this manuscript are protocols for biochemical analysis by thermal shift assay, multi angle light scattering (MALS), and size exclusion chromatography (SEC), which optimize the stability and homogeneity of the sample directed to crystallization trials and so enhance the probability of success. The second part of this procedure describes the characterization of the SBP crystals using a tunable wavelength anomalous diffraction synchrotron beamline, and data collection protocols for measuring data that can be used to resolve the crystallized protein structure.

A streamlined protocol for performing an extensive biochemical and structural characterization of a carbohydrate substrate binding protein from Streptococcus pneumoniae is presented.

탄수화물 전송 기질 바인딩 단백질 SP0092의 생 화 학적 및 구조적 특성

Development of new antimicrobials and vaccines for Streptococcus pneumoniae (pneumococcus) are necessary to halt the rapid rise in multiple resistant ...

Fractionation for Resolution of Soluble and Insoluble Huntingtin Species

The accumulation of misfolded proteins is central to pathology in Huntington's disease (HD) and many other neurodegenerative disorders. Specifically, a key pathological feature of HD is the aberrant accumulation of mutant HTT (mHTT) protein into high molecular weight complexes and intracellular inclusion bodies composed of fragments and other proteins. Conventional methods to measure and understand the contributions of various forms of mHTT-containing aggregates include fluorescence microscopy, western blot analysis, and filter trap assays. 
However, most of these methods are conformation specific, and therefore may not resolve the full state of mHTT protein flux due to the complex nature of aggregate solubility and resolution.
For the identification of aggregated mHTT and various modified forms and complexes, separation and solubilization of the cellular aggregates and fragments is mandatory. Here we describe a method to isolate and visualize soluble mHTT, monomers, oligomers, fragments, and an insoluble high molecular weight (HMW) accumulated mHTT species. HMW mHTT tracks with disease progression, corresponds with mouse behavior readouts, and has been beneficially modulated by certain therapeutic interventions1. This approach can be used with mouse brain, peripheral tissues, and cell culture but may be adapted to other model systems or disease contexts.

A method is described for fractionation of insoluble and soluble mutant huntingtin species from mouse brain and cell culture. The method described is useful for characterization and quantification of huntingtin protein flux and aids in analyzing protein homeostasis in disease pathogenesis and in the presence of perturbations

가용성과 불용 성 Huntingtin의 해상도 대 한 분류

The accumulation of misfolded proteins is central to pathology in Huntington's disease (HD) and many other neurodegenerative disorders. Specifically, a ...

Purification and Reconstitution of TRPV1 for Spectroscopic Analysis

Polymodal ion channels transduce multiple stimuli of different natures into allosteric changes; these dynamic conformations are challenging to determine and remain largely unknown. With recent advances in single-particle cryo-electron microscopy (cryo-EM) shedding light on the structural features of agonist binding sites and the activation mechanism of several ion channels, the stage is set for an in-depth dynamic analysis of their gating mechanisms using spectroscopic approaches. Spectroscopic techniques such as electron paramagnetic resonance (EPR) and double electron-electron resonance (DEER) have been mainly restricted to the study of prokaryotic ion channels that can be purified in large quantities. The requirement for large amounts of functional and stable membrane proteins has hampered the study of mammalian ion channels using these approaches. EPR and DEER offer many advantages, including determination of the structure and dynamic changes of mobile protein regions, albeit at low resolution, that might be difficult to obtain by X-ray crystallography or cryo-EM, and monitoring reversible gating transition (i.e., closed, open, sensitized, and desensitized). Here, we provide protocols for obtaining milligrams of functional detergent-solubilized transient receptor potential cation channel subfamily V member 1 (TRPV1) that can be labeled for EPR and DEER spectroscopy.

This article describes specific methods to obtain biochemical quantities of detergent-solubilized TRPV1 for spectroscopic analysis. The combined protocols provide biochemical and biophysical tools that can be adapted to facilitate structural and functional studies for mammalian ion channels in a membrane-controlled environment.

정화 및 분 광 분석에 대 한 TRPV1의 재구성

Polymodal ion channels transduce multiple stimuli of different natures into allosteric changes; these dynamic conformations are challenging to determine ...

Defining Hsp33's Redox-regulated Chaperone Activity and Mapping Conformational Changes on Hsp33 Using Hydrogen-deuterium Exchange Mass Spectrometry

Living organisms regularly need to cope with fluctuating environments during their life cycle, including changes in temperature, pH, the accumulation of reactive oxygen species, and more. These fluctuations can lead to a widespread protein unfolding, aggregation, and cell death. Therefore, cells have evolved a dynamic and stress-specific network of molecular chaperones, which maintain a "healthy" proteome during stress conditions. ATP-independent chaperones constitute one major class of molecular chaperones, which serve as first-line defense molecules, protecting against protein aggregation in a stress-dependent manner. One feature these chaperones have in common is their ability to utilize structural plasticity for their stress-specific activation, recognition, and release of the misfolded client. 
In this paper, we focus on the functional and structural analysis of one such intrinsically disordered chaperone, the bacterial redox-regulated Hsp33, which protects proteins against aggregation during oxidative stress. Here, we present a toolbox of diverse techniques for studying redox-regulated chaperone activity, as well as for mapping conformational changes of the chaperone, underlying its activity. Specifically, we describe a workflow which includes the preparation of fully reduced and fully oxidized proteins, followed by an analysis of the chaperone anti-aggregation activity in vitro using light-scattering, focusing on the degree of the anti-aggregation activity and its kinetics. To overcome frequent outliers accumulated during aggregation assays, we describe the usage of Kfits, a novel graphical tool which allows easy processing of kinetic measurements. This tool can be easily applied to other types of kinetic measurements for removing outliers and fitting kinetic parameters. To correlate the function with the protein structure, we describe the setup and workflow of a structural mass spectrometry technique, hydrogen-deuterium exchange mass spectrometry, that allows the mapping of conformational changes on the chaperone and substrate during different stages of Hsp33 activity. The same methodology can be applied to other protein-protein and protein-ligand interactions.

One of the most challenging stress conditions that organisms encounter during their lifetime involves the accumulation of oxidants. During oxidative stress, cells heavily rely on molecular chaperones. Here, we present methods used to investigate the redox-regulated anti-aggregation activity, as well as to monitor structural changes governing the chaperone function using HDX-MS.

Hsp33의 Redox 규제 보호자 활동을 정의 하 고 수소 중수소 교환 질량 분석을 사용 하 여 Hsp33에 구조적 변화를 매핑

Living organisms regularly need to cope with fluctuating environments during their life cycle, including changes in temperature, pH, the accumulation of ...

Sampling and Pretreatment of Tooth Enamel Carbonate for Stable Carbon and Oxygen Isotope Analysis

Stable carbon and oxygen isotope analysis of human and animal tooth enamel carbonate has been applied in paleodietary, paleoecological, and paleoenvironmental research from recent historical periods back to over 10 million years ago. Bulk approaches provide a representative sample for the period of enamel mineralization, while sequential samples within a tooth can track dietary and environmental changes during this period. While these methodologies have been widely applied and described in archaeology, ecology, and paleontology, there have been no explicit guidelines to aid in the selection of necessary lab equipment and to thoroughly describe detailed laboratory sampling and protocols. In this article, we document textually and visually, the entire process from sampling through pretreatment and diagenetic screening to make the methodology more widely available to researchers considering its application in a variety of laboratory settings.

Stable carbon and oxygen isotope analysis of human and animal tooth enamel carbonate has been used as a proxy for individual diet and environmental reconstruction. Here, we provide a detailed description and visual documentation of bulk and sequential tooth enamel sampling as well as pretreatment of archaeological and paleontological samples.

안정적인 탄소 및 산소 동위 원소 분석을 위한 샘플링 및 치아에 나 멜의 전처리 탄산염

Stable carbon and oxygen isotope analysis of human and animal tooth enamel carbonate has been applied in paleodietary, paleoecological, and ...

Detection of Phospholipase C Activity in the Brain Homogenate from the Honeybee

The honeybee is a model organism for evaluating complex behaviors and higher brain function, such as learning, memory, and division of labor. The mushroom body (MB) is a higher brain center proposed to be the neural substrate of complex honeybee behaviors. Although previous studies identified genes and proteins that are differentially expressed in the MBs and other brain regions, the activities of the proteins in each region are not yet fully understood. To reveal the functions of these proteins in the brain, pharmacologic analysis is a feasible approach, but it is first necessary to confirm that pharmacologic manipulations indeed alter the protein activity in these brain regions.
We previously identified a higher expression of genes encoding phospholipase C (PLC) in the MBs than in other brain regions, and pharmacologically assessed the involvement of PLC in honeybee behavior. In that study, we biochemically tested two pharmacologic agents and confirmed that they decreased PLC activity in the MBs and other brain regions. Here, we present a detailed description of how to detect PLC activity in honeybee brain homogenate. In this assay system, homogenates derived from different brain regions are reacted with a synthetic fluorogenic substrate, and fluorescence resulting from PLC activity is quantified and compared between brain regions. We also describe our evaluation of the inhibitory effects of certain drugs on PLC activity using the same system. Although this system is likely affected by other endogenous fluorescence compounds and/or the absorbance of the assay components and tissues, the measurement of PLC activity using this system is safer and easier than that using the traditional assay, which requires radiolabeled substrates. The simple procedure and manipulations allow us to examine PLC activity in the brains and other tissues of honeybees involved in different social tasks.

To test the inhibitory effects of pharmacologic agents on phospholipase C (PLC) in different regions of the honeybee brain, we present a biochemical assay to measure PLC activity in those regions. This assay could be useful for comparing PLC activity among tissues, as well as among bees exhibiting different behaviors.

꿀벌에서 두뇌 Homogenate에서 Phospholipase C 활동의 탐지

The honeybee is a model organism for evaluating complex behaviors and higher brain function, such as learning, memory, and division of labor. The mushroom ...

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.

측정 및 해석의 전체 산소 소비 속도 비행 머리 세그먼트

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

Fully Processed Recombinant KRAS4b: Isolating and Characterizing the Farnesylated and Methylated Protein

Protein prenylation is a key modification that is responsible for targeting proteins to intracellular membranes. KRAS4b, which is mutated in 22% of human cancers, is processed by farnesylation and carboxymethylation due to the presence of a &#39;CAAX&#39; box motif at the C-terminus. An engineered baculovirus system was used to express farnesylated and carboxymethylated KRAS4b in insect cells and has been described previously. Here, we describe the detailed, practical purification and biochemical characterization of the protein. Specifically, affinity and ion exchange chromatography were used to purify the protein to homogeneity. Intact and native mass spectrometry was used to validate the correct modification of KRAS4b and to verify nucleotide binding. Finally, membrane association of farnesylated and carboxymethylated KRAS4b to liposomes was measured using surface plasmon resonance spectroscopy.

Prenylation is an important modification on peripheral membrane binding proteins. Insect cells can be manipulated to produce farnesylated and carboxymethylated KRAS4b in quantities that enable biophysical measurements of protein-protein and protein-lipid interactions

완전히 가공된 재조합 KRAS4b: 판자및 메틸화 단백질을 분리하고 특성화

Protein prenylation is a key modification that is responsible for targeting proteins to intracellular membranes. KRAS4b, which is mutated in 22% of human ...

Extraction and Purification of FAHD1 Protein from Swine Kidney and Mouse Liver

Fumarylacetoacetate hydrolase domain-containing protein 1 (FAHD1) is the first identified member of the FAH superfamily in eukaryotes, acting as oxaloacetate decarboxylase in mitochondria. This article presents a series of methods for the extraction and purification of FAHD1 from swine kidney and mouse liver. Covered methods are ionic exchange chromatography with fast protein liquid chromatography (FPLC), preparative and analytical gel filtration with FPLC, and proteomic approaches. After total protein extraction, ammonium sulfate precipitation and ionic exchange chromatography were explored, and FAHD1 was extracted via a sequential strategy using ionic exchange and size-exclusion chromatography. This representative approach may be adapted to other proteins of interest (expressed at significant levels) and modified for other tissues. Purified protein from tissue may support the development of high-quality antibodies, and/or potent and specific pharmacological inhibitors.

This protocol describes how to extract fumarylacetoacetate hydrolase domain-containing protein 1 (FAHD1) from swine kidney and mouse liver. The listed methods may be adapted to other proteins of interest and modified for other tissues.