Name: defining substrate specificities for lipase and phospholipase candidates
Uploaded: 2016-11-23T12:30:00.000+00:00
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Description: Watch this Scientific Journal Video about Defining Substrate Specificities for Lipase and Phospholipase Candidates at JoVE.com

This work was supported by grants from Consejo Nacional de Ciencias y Tecnolog&#237;a-M&#233;xico (CONACyT-Mexico) (82614, 153998, 253549, and 178359&#160;in Investigaci&#243;n Cient&#237;fica B&#225;sica as well as 118 in Investigaci&#243;n en Fronteras de la Ciencia) and from Direcci&#243;n General de Asuntos de Personal Acad&#233;mico-Universidad Nacional Aut&#243;noma de M&#233;xico (DGAPA-UNAM; PAPIIT IN202616, IN203612).

<ol>
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USA. 107 (1), 302-307 (2010).</li><li>Pech-Canul, A., 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=FadD+is+required+for+utilization+of+endogenous+fatty+acids+released+from+membrane+lipids.">FadD is required for utilization of endogenous fatty acids released from membrane lipids.</a> J. Bacteriol. 193 (22), 6295-6304 (2011).</li><li>Banerji, S., Flieger, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Patatin-like+proteins:+a+new+family+of+lipolytic+enzymes+present+in+bacteria?.">Patatin-like proteins: a new family of lipolytic enzymes present in bacteria?.</a> Microbiology. 150 (Pt 3), 522-525 (2004).</li><li>Sahonero-Canavesi, D. X., 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=Fatty+acid-releasing+activities+in+Sinorhizobium+meliloti+include+unusual+diacylglycerol+lipase.">Fatty acid-releasing activities in Sinorhizobium meliloti include unusual diacylglycerol lipase.</a> Environ. Microbiol. 17 (9), 3391-3406 (2015).</li><li>Fischer, M., Pleiss, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+Lipase+Engineering+Database:+a+navigation+and+analysis+tool+for+protein+families.">The Lipase Engineering Database: a navigation and analysis tool for protein families.</a> Nucl. Acid. Res. 31 (1), 319-321 (2003).</li><li>Sigrist, C. J. A., 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=New+and+continuing+developments+at+PROSITE.">New and continuing developments at PROSITE.</a> Nucleic Acids Res. 41 (Database issue), D344-D347 (2013).</li><li>Lorenz, T. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Polymerase+Chain+Reaction:+Basic+Protocol+Plus+Troubleshooting+and+Optimization+Strategies.">Polymerase Chain Reaction: Basic Protocol Plus Troubleshooting and Optimization Strategies.</a> J. Vis. Exp. (63), e3998 (2012).</li><li>Untergasser, A., 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=Primer3+-+new+capabilities+and+interfaces.">Primer3 - new capabilities and interfaces.</a> Nucl. Acids Res. 40 (15), e115 (2012).</li><li>Studier, F. W., Rosenberg, A. H., Dunn, J. J., Dubendorff, J. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Use+of+T7+RNA+polymerase+to+direct+expression+of+cloned+genes.">Use of T7 RNA polymerase to direct expression of cloned genes.</a> Methods Enzymol. 185, 60-89 (1990).</li><li>Miller, J. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Experiments in Molecular Genetics. , (1972).</li><li>Desjardins, P., Hansen, J. B., Allen, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microvolume+Protein+Concentration+Determination+using+the+NanoDrop+2000c+Spectrophotometer.">Microvolume Protein Concentration Determination using the NanoDrop 2000c Spectrophotometer.</a> J. Vis. Exp. (33), e1610 (2009).</li><li>Bligh, E. G., Dyer, W. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+rapid+method+for+total+lipid+extraction+and+purification.">A rapid method for total lipid extraction and purification.</a> Can. J. Biochem. 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. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Kinome+profiling.">Kinome profiling.</a> Scientifica (Cairo). , (2012).</li><li>Manafi, M., Kneifel, W., Bascomb, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fluorogenic+and+chromogenic+substrates+used+in+bacterial+diagnostics.">Fluorogenic and chromogenic substrates used in bacterial diagnostics.</a> Microbiol. Rev. 55 (3), 335-348 (1991).</li><li>Kuznetsova, 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=Enzyme+genomics:+Application+of+general+enzymatic+screens+to+discover+new+enzymes.">Enzyme genomics: Application of general enzymatic screens to discover new enzymes.</a> FEMS Microbiol. Rev. 29 (2), 263-279 (2005).</li><li>Scopes, R. 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The authors have nothing to disclose.

Over the past 20 years, genomes of many organisms have been sequenced and although a wealth of genome sequence data has been generated, functional interpretation is lagging behind and therefore hampers our understanding of genome function. Gene functions in genomes are often assigned based on similarity to genes of known function or occurrence of conserved motifs. However, the precise function of a given gene is often not known. Especially, predicted structural genes for enzymes cannot be easily explored by omic techniqu...

Glycerol-based lipids such as triacylglycerols and (glycero)phospholipids constitute important and probably the best-known lipid classes1. Triacylglycerols (TAGs) are fats or oils, which usually function as storage lipids, and therefore as potential energy and carbon sources. TAGs can be degraded by lipases, which are frequently secreted by the producing organism to digest external TAGs and make them available as carbon sources. Also, lipases have been widely studied over the years due to their important biotechnological applications2.
Due to their amphiphilic nature and their near-cylindric shape, (glycero)phospholipids exhibit membrane-forming properties and usually constitute the major lipidic components of a bilayered membrane3. In simple microorganisms, such as the bacterium Escherichia coli, only three major head group variants, phosphatidylglycerol (PG), cardiolipin (CL), and phosphatidylethanolamine (PE) are encountered, although one should be aware that each one of them can be substituted with a considerable number of different fatty acyl chains at the sn-1 or sn-2 position giving rise to a large number of different molecular species4. Other bacteria might have other phospholipids in addition or instead. For example, Sinorhizobium meliloti, a soil bacterium, which is able to form a nitrogen-fixing root nodule symbiosis with the legume alfalfa (Medicago sativa), contains in addition to PE a second zwitterionic phospholipid, phosphatidylcholine (PC)5. Also, lipids not containing phosphorus or glycerol might be amphiphilic and form part of the cellular membrane. For example, upon phosphorus-limiting growth conditions, in S. meliloti, (glycero)phospholipids are largely replaced by membrane lipids that do not contain phosphorus, i.e., sulfolipids, ornithine lipids, and diacylglyceryl trimethylhomoserine (DGTS)6. In bacteria, DGTS is formed from diacylglycerol (DAG) in a two-step pathway7 but the source for DAG generation was not clear. Pulse-chase experiments suggested that PC might be a precursor for DGTS8 and using the methodology described in this manuscript we could identify a phospholipase C (PlcP, SMc00171) that is formed under phosphorus-limiting conditions and which can convert PC into DAG and phosphocholine8.
In a separate study, we discovered that an acyl-CoA synthetase (FadD)-deficient mutant of S. meliloti or of Escherichia coli accumulated free fatty acids when entering stationary phase of growth9. Although these fatty acids seemed to be derived from membrane lipids, the precise source for the free fatty acids or the enzyme(s) liberating them were not known. Again, employing the strategy outlined in this manuscript, two patatin-like10 (phospho)lipases (SMc00930 and SMc01003) that contributed to the formation of free fatty acids in S. meliloti11 were predicted. Surprisingly, SMc01003 used DAG as substrate converting it to monoacylglycerol and finally glycerol and free fatty acids11. Therefore, SMc01003 is a DAG lipase (DglA).
Although a number of algorithms exist for predicting potential (phospho)lipases12,13, their precise function and physiological role is usually not known. Here we outline a protocol, to clone and overexpress predicted or potential (phospho)lipases. This manuscript explains how enzyme assays can be developed and optimized for the overexpressed (phospho)lipase by using artificial chromogenic substrates. We provide examples how with an optimized enzyme assay the real (phospho)lipase substrate can be encountered and how these findings might enrich our understanding of microbial physiology.

<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

Microorganisms produce a wide spectrum of (phospho)lipases that are secreted in order to make external substrates available for the organism. Alternatively, other (phospho)lipases may be physically associated with the producing organism causing a turnover of intrinsic lipids and frequently giving rise to a remodeling of the cellular membranes. Although potential (phospho)lipases can be predicted with a number of algorithms when the gene/protein sequence is available, experimental proof of the enzyme activities, substrate specificities, and potential physiological functions has frequently not been obtained. This manuscript describes the optimization of assay conditions for prospective (phospho)lipases with unknown substrate specificities and how to employ these optimized conditions in the search for the natural substrate of a respective (phospho)lipase. Using artificial chromogenic substrates, such as p-nitrophenyl derivatives, may help to detect a minor enzymatic activity for a predicted (phospho)lipase under standard conditions. Having encountered such a minor enzymatic activity, the distinct parameters of an enzyme assay can be varied in order to obtain a more efficient hydrolysis of the artificial substrate. After having determined the conditions under which an enzyme works well, a variety of potential natural substrates should be assayed for their degradation, a process that can be followed employing distinct chromatographic methods. The definition of substrate specificities for new enzymes, often provides hypotheses for a potential physiological role of these enzymes, which then can be tested experimentally. Following these guidelines, we were able to identify a phospholipase C (SMc00171) that degrades phosphatidylcholine to phosphocholine and diacylglycerol, in a crucial step for the remodeling of membranes in the bacterium Sinorhizobium meliloti upon phosphorus-limiting conditions of growth. For two predicted patatin-like phospholipases (SMc00930 and SMc01003) of the same organism, we could redefine their substrate specificities and clarify that SMc01003 is a diacylglycerol lipase.

Activity of PC-specific Phospholipase C SMc00171 with Bis-p-nitrophenyl Phosphate
Cell-free extracts obtained from E. coli BL21(DE3) x pLysS, which had smc00171 expressed, were studied for their ability to hydrolyze bis-p-nitrophenyl phosphate esters, using a spectrophotometric enzymatic assay, measuring the p-NP formed. No hydrolytic...

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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.

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 San Sebastian

Research

JoVE Journal

Biochemistry

14.9K Views.  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.

Video: 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.

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

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.

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

Chemistry

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.

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.

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.

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.

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.

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

Bioengineering

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.

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.

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

Defining Substrate Specificities for Lipase and Phospholipase Candidates

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Chapters in this video

A Fluorescence-based Assay of Phospholipid Scramblase Activity

Unraveling Entropic Rate Acceleration Induced by Solvent Dynamics in Membrane Enzymes

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

Use of Microscale Thermophoresis to Measure Protein-Lipid Interactions

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

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

Analysis of Lipid Signaling in Drosophila Photoreceptors using Mass Spectrometry

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

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

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