Name: electrochemical detection of deuterium kinetic isotope effect on extracellular electron transport in shewanella oneidensis mr-1
Uploaded: 2018-04-16T14:30:00
Description: Watch this Scientific Journal Video about Electrochemical Detection of Deuterium Kinetic Isotope Effect on Extracellular Electron Transport in Shewanella oneidensis MR-1 at JoVE.com

This work was financially supported by a Grant-in-Aid for Specially Promoted Research from the Japan Society for Promotion of Science (JSPS) KAKENHI Grant Number 24000010, 17H04969, and JP17J02602, the US Office of Naval Research Global (N62909-17-1-2038). Y.T. is a JSPS Research Fellow and supported by JSPS through the Program for Leading Graduate Schools (MERIT).

1. Formation of a Monolayer Biofilm of S. oneidensis MR-1 on an ITO Electrode (Figure 1)
NOTE: To prevent the contamination of the electrochemical reactor with other microbes, all the media, implements, and components of the electrochemical reactor should be sterilized in advance. When using S. oneidensis MR-1 cells and constructing the electrochemical reactors, all the procedures should be conducted on a clean bench.
<ol>
	<li>Cultivation of <...

<ol>
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T., 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=Iron+corrosion+by+novel+anaerobic+microorganisms.">Iron corrosion by novel anaerobic microorganisms.</a> Nature. 427 (6977), 829-832 (2004).</li><li>McGlynn, S. E., Chadwick, G. L., Kempes, C. P., Orphan, V. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Single+cell+activity+reveals+direct+electron+transfer+in+methanotrophic+consortia.">Single cell activity reveals direct electron transfer in methanotrophic consortia.</a> Nature. 526 (7574), 531-535 (2015).</li><li>Okamoto, A., Nakamura, R., Nealson, K. H., Hashimoto, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Bound+Flavin+Model+Suggests+Similar+Electron-Transfer+Mechanisms+in+Shewanella+and+Geobacter.">Bound Flavin Model Suggests Similar Electron-Transfer Mechanisms in Shewanella and Geobacter.</a> Chemelectrochem. 1 (11), 1808-1812 (2014).</li><li>Okamoto, A., Hashimoto, K., Nealson, K. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Flavin+Redox+Bifurcation+as+a+Mechanism+for+Controlling+the+Direction+of+Electron+Flow+during+Extracellular+Electron+Transfer.">Flavin Redox Bifurcation as a Mechanism for Controlling the Direction of Electron Flow during Extracellular Electron Transfer.</a> Angew. Chem. Int. 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Our whole-cell electrochemical assay has several technical advantages compared with protein electrochemistry. While protein purification requires multi-step time-consuming procedures, our whole-cell method takes one day of self-organized biofilm formation after cell culture. To achieve a stable interaction between OM c-Cyts and the electrode, we need only sterilization and cleaning of the electrode surface; it does not require electrode modification for organizing the orientation of proteins4</...

Electrochemical techniques to directly characterize a redox protein in an intact bacterial cell have recently emerged since the discovery of metal-reducing microbial strains, such as S. oneidensis MR-1 or Geobacter sulfurreducens PCA, which have outer membrane c-type cytochrome complexes (OM c-Cyts) exposed to the cell exterior1,2,3,4,5. The OM c-Cyts mediate electron transport from the respiratory chain to solid substrates located extracellularly. This transport is referred to as extracellular electron transport (EET)1,6 and is a critical process for emerging biotechnologies, such as microbial fuel cells6. Therefore, to understand the underlying EET kinetics and mechanisms, and its link to microbial physiology, OM c-Cyts have been investigated using whole-cell electrochemistry4,7, combined with microscopy8,9, spectroscopy10,11, and molecular biology2,4. In contrast, methods to investigate the impact of EET-associated cation transport, e.g., protons, on EET kinetics in living cells have been scarcely established, despite proton transport across bacterial membranes having a critical role in signaling, homeostasis, and energy production12,13,14. In the present study, we describe a technique to examine the impact of proton transport on EET kinetics in the S. oneidensis MR-1 cell using whole-cell electrochemical measurements, which requires the identification of the rate-limiting step in microbial current production15.
One direct way to evaluate the contribution of proton transport on the associated EET is the deuterium kinetic isotope effect (KIE). The KIE is observable as the change in electron transfer kinetics upon the replacement of protons with deuterium ions, which represents the impact of proton transport on electron transfer kinetics16. The theory of KIE itself has been well established using electrochemical measurements with purified enzymes17. However, since current production in S. oneidensis MR-1 results from multiple, diverse, and fluctuating processes18, one cannot simply identify EET as the rate-limiting process. To observe the KIE on proton transport processes coupled with EET, we need to confirm that the microbial current production is limited by electron transport via OM c-Cyts to the electrode. For this purpose, we replaced the supernatant solution with fresh medium containing a high concentration of lactate as an electron donor at the optimal pH and temperature conditions before KIE measurement; this replacement served two roles: (1) it enhanced the rate of the upstream metabolic processes compared to the EET, and (2) omitted the swimming cells in the supernatant released from the monolayer biofilm of S. oneidensis MR-1 on the working electrode (indium tin-doped oxide (ITO) electrode). The presented detailed protocol is intended to help new practitioners maintain and confirm that the EET process is the rate-determining step.

<table>
	<tbody>
		<tr>
			<td>Glass cylinder</td>
			<td>N/A</td>
			<td>N/A</td>
			<td>Custom-made, used as the electrochemical reactor</td>
		</tr>
		<tr>
			<td>PTFE cover and base</td>
			<td>N/A</td>
			<td>N/A</td>
			<td>Custom-made, used as a cover and a foundation of the electrochemical reactor</td>
		</tr>
		<tr>
			<td>Buthyl rubber</td>
			<td>N/A</td>
			<td>N/A</td>
			<td>Custom-made, inserted between each component of electrochemical reactor</td>
		</tr>
		<tr>
			<td>Septa</td>
			<td>GL Science</td>
			<td>3007-16101</td>
			<td>Used as an injection port of electrochemical reactor</td>
		</tr>
		<tr>
			<td>Indium tin-doped oxide (ITO) electrode</td>
			<td>GEOMATEC</td>
			<td>No.0001</td>
			<td>Used as a working electrode, 5&#937;/sq</td>
		</tr>
		<tr>
			<td>Ag/AgCl KCl saturated electrode</td>
			<td>HOKUTO DENKO</td>
			<td>HX-R5</td>
			<td>Used as a reference electrode, &#934;0.30mm</td>
		</tr>
		<tr>
			<td>Platinum wire</td>
			<td>The Nilaco Cooporation</td>
			<td>PT-351325</td>
			<td>Used as a counter electrode</td>
		</tr>
		<tr>
			<td>Luria-Bertani (LB) Broth, Miller</td>
			<td>Becton, Dichkinson and Company</td>
			<td>244620</td>
			<td>Medium for precultivation of S. oneidensis MR-1</td>
		</tr>
		<tr>
			<td>Bacto agar</td>
			<td>Becton, Dichkinson and Company</td>
			<td>214010</td>
			<td></td>
		</tr>
		<tr>
			<td>Anthraquinone-1-sulfonate (&#945;-AQS)</td>
			<td>TCI</td>
			<td>A1428</td>
			<td></td>
		</tr>
		<tr>
			<td>Flavin mononucleotide (FMN)</td>
			<td>Wako</td>
			<td>184-00831</td>
			<td></td>
		</tr>
		<tr>
			<td>NaHCO3</td>
			<td>Wako</td>
			<td>191-01305</td>
			<td>Used for defined medium (DM)</td>
		</tr>
		<tr>
			<td>CaCl2 &#183; 2H2O</td>
			<td>Wako</td>
			<td>031-00435</td>
			<td>Used for DM</td>
		</tr>
		<tr>
			<td>NH4Cl</td>
			<td>Wako</td>
			<td>011-03015</td>
			<td>Used for DM</td>
		</tr>
		<tr>
			<td>MgCl2 &#183; 6H2O</td>
			<td>Wako</td>
			<td>135-00165</td>
			<td>Used for DM</td>
		</tr>
		<tr>
			<td>NaCl</td>
			<td>Wako</td>
			<td>191-01665</td>
			<td>Used for DM</td>
		</tr>
		<tr>
			<td>2-[4-(2-hydroxyethyl)-1-piperazinyl] ethanesulfonic acid (HEPES)</td>
			<td>DOJINDO</td>
			<td>346-08235</td>
			<td>Used for DM</td>
		</tr>
		<tr>
			<td>Sodium Lactate Solution</td>
			<td>Wako</td>
			<td>195-02305</td>
			<td></td>
		</tr>
		<tr>
			<td>Bacto Yeast Extract</td>
			<td>Becton, Dichkinson and Company</td>
			<td>212750</td>
			<td></td>
		</tr>
		<tr>
			<td>Deuterium oxide (D, 99.9%)</td>
			<td>Cambridge Isotope Laboratories, Inc.</td>
			<td>DLM-4-PK</td>
			<td>Additive for kinetic isotope effect experiments</td>
		</tr>
		<tr>
			<td>Incubator</td>
			<td>TOKYO RIKAKIKAI CO. LTD.</td>
			<td>LTI-601SD</td>
			<td>Used for precultivation</td>
		</tr>
		<tr>
			<td>Shaker</td>
			<td>TAITEC</td>
			<td>NR-3</td>
			<td>Used for precultivation</td>
		</tr>
		<tr>
			<td>Autoclave machine</td>
			<td>TOMY SEIKO CO. LTD.</td>
			<td>LSX-500</td>
			<td>Used for sterilization of the electrochemical reactor and the medium</td>
		</tr>
		<tr>
			<td>Clean bench</td>
			<td>SANYO</td>
			<td>MCV-91BNF</td>
			<td>Used to prevent the contamination of the electrochemical reactor and the medium with other microbes</td>
		</tr>
		<tr>
			<td>Centrifuge separator</td>
			<td>Eppendorf</td>
			<td>5430R</td>
			<td>Rotational speed upto 6000&#215;g is required</td>
		</tr>
		<tr>
			<td>Nitrogen gas generator</td>
			<td>Puequ CO. LTD.</td>
			<td>PNTN-2</td>
			<td>Nitrogen gas cylinder can also be used instead of gas generator</td>
		</tr>
		<tr>
			<td>UV-vis spectrometer</td>
			<td>SHIMADZU</td>
			<td>UV-1800</td>
			<td>Used for optimization of cell density</td>
		</tr>
		<tr>
			<td>Potentiostat</td>
			<td>BioLogic</td>
			<td>VMP3</td>
			<td>Used for biofilm formation and kinetic isotope effect experiments</td>
		</tr>
		<tr>
			<td>Thermal water circulator</td>
			<td>AS ONE</td>
			<td>TR-1A</td>
			<td>Used for maintanance of temperature of electrochemcial reactor</td>
		</tr>
		<tr>
			<td>Faraday cage</td>
			<td>HOKUTO DENKO</td>
			<td>HS-201S</td>
			<td>Used for electrochemical experiments</td>
		</tr>
	</tbody>
</table>

electrochemical detection of deuterium kinetic isotope effect on extracellular electron transport in shewanella oneidensis mr-1

Direct electrochemical detection of c-type cytochrome complexes embedded in the bacterial outer membrane (outer membrane c-type cytochrome complexes; OM c-Cyts) has recently emerged as a novel whole-cell analytical method to characterize the bacterial electron transport from the respiratory chain to the cell exterior, referred to as the extracellular electron transport (EET). While the pathway and kinetics of the electron flow during the EET reaction have been investigated, a whole-cell electrochemical method to examine the impact of cation transport associated with EET has not yet been established. In the present study, an example of a biochemical technique to examine the deuterium kinetic isotope effect (KIE) on EET through OM c-Cyts using a model microbe, Shewanella oneidensis MR-1, is described. The KIE on the EET process can be obtained if the EET through OM c-Cyts acts as the rate-limiting step in the microbial current production. To that end, before the addition of D2O, the supernatant solution was replaced with fresh media containing a sufficient amount of the electron donor to support the rate of upstream metabolic reactions, and to remove the planktonic cells from a uniform monolayer biofilm on the working electrode. Alternative methods to confirm the rate-limiting step in microbial current production as EET through OM c-Cyts are also described. Our technique of a whole-cell electrochemical assay for investigating proton transport kinetics can be applied to other electroactive microbial strains.

After 25 h of potential application at +0.4 V (versus SHE), a monolayer biofilm was formed on the working electrode of ITO glass, which was previously confirmed by either a scanning electron microscopy or a confocal microscopy4. The representative time course of current production from the S. oneidensis MR-1 during the formation of a monolayer biofilm is shown in Figure 2. Although the current alters in every measurement, the ...

Watch this Scientific Journal Video about Electrochemical Detection of Deuterium Kinetic Isotope Effect on Extracellular Electron Transport in Shewanella oneidensis MR-1 at JoVE.com

Electrochemical Detection of Deuterium Kinetic Isotope Effect on Extracellular Electron Transport in Shewanella oneidensis MR-1

Here we present a protocol of whole-cell electrochemical experiments to study the contribution of proton transport to the rate of extracellular electron transport via the outer-membrane cytochromes complex in Shewanella oneidensis MR-1.

Electrochemical Detection of Deuterium Kinetic Isotope Effect on Extracellular Electron Transport in Shewanella oneidensis MR-1

Direct electrochemical detection of c-type cytochrome complexes embedded in the bacterial outer membrane (outer membrane c-type cytochrome complexes; OM ...

The overall goal of this protocol is to observe the contribution of proton transport to the rate of bacterial extracellular electron transport by the detection of the deuterium kinetic isotope effect. Our method reveals the impact of proton transport on the kinetics of extracellular electron transport or EET from bacteria to an electrode via outer membrane c-type cytochromes. Our assay reflects the native proton management in EET because we used living bacteria rather than purified enzyme.However, our challenge was to remove the noises from other cellular processes. The deuterium kinetic isotope effect termed as KIE is one of the best technique to examine the contribution of proton transport on the associated electron transport process. To grow Shewanella MR-1 cells, transfer one colony of the cells grown on an agar plate into 15 milliliters of LB medium.Grow the cells at 30 degrees Celsius for 24 hours in aerobic conditions with shaking at 160 rpm. Centrifuge the cell suspension at 6, 000 times gravity for 10 minutes. Then, resuspend the resultant cell pellet in 15 milliliters of Defined Medium, supplemented with 10 millimolar lactate as the source of carbon and 0.5 grams per liter yeast extract as trace elements for the cells.After further cultivating the cell suspension aerobically at 30 degrees Celsius for 12 hours with shaking at 160 rpm, centrifuge again at 6, 000 times gravity for 10 minutes. Wash the resultant cell pellet twice with DM medium by centrifugation for 10 minutes at 6, 000 times gravity before the electrochemical experiment. To construct a three-electrode electrochemical reactor, put an ITO substrate as the working electrode at the bottom of the reactor.Subsequently, insert a glass cylinder and a polytetrafluoroethylene cover and insert a platinum wire as the counter-electrode. Then, insert a silver chloride electrode into the reactor as the reference electrode. Next, add four milliliters of DM supplemented with 10 millimolar lactate and 0.5 grams per liter yeast extract into the electrochemical reactor.After confirming that there is no leakage from the electrochemical reactor, flow the nitrogen gas into the electrochemical reactor over 20 minutes to maintain anerobic conditions inside the electrochemical reactor. Connect the electrochemical reactor to a potentiostat and apply a positive 0.4 volts to the ITO electrode, keeping the temperature of the electrochemical reactor at 30 degrees Celsius using an external water circulation system. To perform electrochemical cultivation, first adjust the cell density of the suspension to an optical density of 1.43 at 600 nanometers with DM supplemented by 10 millimolar lactate and 0.5 grams per liter yeast extract.Add 0.3 milliliters of the cell suspension into the electrochemical reactor through the injection port using a syringe. The OD 600 in the reactor changes to 0.1. Continue the potential application at positive 0.4 volts to the ITO electrode for 25 hours.Stop the potential application and disconnect the electrochemical reactor from the potentiostat and the water circulation system. Flow the nitrogen gas into a bottle containing DM with 10 millimolar lactate over 20 minutes to remove oxygen from the medium. Now, extremely slowly and gently, remove all the supernatant from inside the electrochemical reactor using a syringe under flowing nitrogen gas.Then, add four milliliters of fresh DM containing 10 millimolar lactate using a syringe. Slant the electrochemical reactor to remove all of the supernatant on the wall of the reactor. Repeat these steps three times in total.Stop the gas flow and connect the electrochemical reactor to the potentiostat again, applying a positive 0.4 volts to the ITO electrode at 303 Kelvin. Confirm that the current production from a monolayer biofilm of Shewanella MR-1 is stable and does not increase rapidly. If the current increases steeply, wait until the current stabilizes with a 5%increase over 10 minutes.Extremely slowly and gently, add 40 microliters of anoxic 50 volume percent D2O into the electrochemical reactor using a syringe such that the concentration is 0.5 volume percent D2O in the reactor. To prevent damage to the biofilm by D20 addition, inject the D2O dropwise. Wait for the current to stabilize and subsequently add the D2O up to 4.4 volume percent to obtain the KIE value, which is the ratio of current production and presence of D2O and H2O.Check the effect of the same volume of H2O addition on the current production. Here, the solid line is the representative result for the microbial current change induced by D2O addition. Addition of D2O sharply decreased the microbial current within 10 seconds, while H20 had almost no current decrease as shown with the dotted line.To confirm that the observed current decrease by D2O is attributable to electron transport through outer membrane cytochromes, a diffusing electron mediator was used. Here addition of 100 micromolar alpha-AQS enhanced the current production by more than five times and the current production remained unaffected by D2O addition. These results indicate that the kinetics of the metabolic reactions are fast enough so that the extracellular electron transfer process is rate-limiting.An alternative method for assessing this is the addition of the flavin molecule into the electrochemical reactor. An instant anodic current increase by flavin addition indicates rate-limitation by the electron transport process via outer membrane cytochromes. After watching this video, you should have a good understanding of how to examine the kinetic isotope effect on the extracellular electron transport process.The most critical point to successfully obtain the kinetic isotope effect is to clarify and confirm the condition where the electron transport through the outer membrane cytochromes limits the rate of current production. In this video, we introduced two methods to confirm this condition, observation of current change by addition of flavin and by addition of diffusing extra mediator, such as alpha-AQS. Once the rate-limitation is confirmed, this system can be used not only for the deuterium experiment but also for the biochemical characterization of outer membrane cytochromes.Furthermore, this protocol could be applicable to examining other electroactive microbes, as long the extracellular electron transport process is rate-limiting. Our methodology provides a basic and general technique to investigate the extracellular electron transport process via outer membrane cytochromes in vivo.

electrochemical-detection-deuterium-kinetic-isotope-effect-on

Research

JoVE Journal

Biochemistry

Reconstitution of Nucleosomes with Differentially Isotope-labeled Sister Histones

Asymmetrically modified nucleosomes contain the two copies of a histone (sister histones) decorated with distinct sets of Post-translational Modifications (PTMs). They are newly identified species with unknown means of establishment and functional implications. Current analytical methods are inadequate to detect the copy-specific occurrence of PTMs on the nucleosomal sister histones. This protocol presents a biochemical method for the in vitro reconstitution of nucleosomes containing differentially isotope-labeled sister histones. The generated complex can be also asymmetrically modified, after including a premodified histone pool during refolding of histone subcomplexes. These asymmetric nucleosome preparations can be readily reacted with histone-modifying enzymes to study modification cross-talk mechanisms imposed by the asymmetrically pre-incorporated PTM using nuclear magnetic resonance (NMR) spectroscopy. Particularly, the modification reactions in real-time can be mapped independently on the two sister histones by performing different types of NMR correlation experiments, tailored for the respective isotope type. This methodology provides the means to study crosstalk mechanisms that contribute to the formation and propagation of asymmetric PTM patterns on nucleosomal complexes.

This protocol describes the reconstitution of nucleosomes containing differentially isotope-labeled sister histones. At the same time, asymmetrically post-translationally modified nucleosomes can be generated after using a premodified histone copy. These preparations can be readily used to study modification crosstalk mechanisms, simultaneously on both sister histones, by using high-resolution NMR spectroscopy.

Asymmetrically modified nucleosomes contain the two copies of a histone (sister histones) decorated with distinct sets of Post-translational Modifications ...

Time-resolved ElectroSpray Ionization Hydrogen-deuterium Exchange Mass Spectrometry for Studying Protein Structure and Dynamics

Intrinsically disordered proteins (IDPs) have long been a challenge to structural biologists due to their lack of stable secondary structure elements. Hydrogen-Deuterium Exchange (HDX) measured at rapid time scales is uniquely suited to detect structures and hydrogen bonding networks that are briefly populated, allowing for the characterization of transient conformers in native ensembles. Coupling of HDX to mass spectrometry offers several key advantages, including high sensitivity, low sample consumption and no restriction on protein size. This technique has advanced greatly in the last several decades, including the ability to monitor HDX labeling times on the millisecond time scale. In addition, by incorporating the HDX workflow onto a microfluidic platform housing an acidic protease microreactor, we are able to localize dynamic properties at the peptide level. In this study, Time-Resolved ElectroSpray Ionization Mass Spectrometry (TRESI-MS) coupled to HDX was used to provide a detailed picture of residual structure in the tau protein, as well as the conformational shifts induced upon hyperphosphorylation.

Conformational flexibility plays a critical role in protein function. Herein, we describe the use of time-resolved electrospray ionization mass spectrometry coupled to hydrogen-deuterium exchange for probing the rapid structural changes that drive function in ordered and disordered proteins.

Intrinsically disordered proteins (IDPs) have long been a challenge to structural biologists due to their lack of stable secondary structure elements. ...

Analyzing Supercomplexes of the Mitochondrial Electron Transport Chain with Native Electrophoresis, In-gel Assays, and Electroelution

The mitochondrial electron transport chain (ETC) transduces the energy derived from the breakdown of various fuels into the bioenergetic currency of the cell, ATP. The ETC is composed of 5 massive protein complexes, which also assemble into supercomplexes called respirasomes (C-I, C-III, and C-IV) and synthasomes (C-V) that increase the efficiency of electron transport and ATP production. Various methods have been used for over 50 years to measure ETC function, but these protocols do not provide information on the assembly of individual complexes and supercomplexes. This protocol describes the technique of native gel polyacrylamide gel electrophoresis (PAGE), a method that was modified more than 20 years ago to study ETC complex structure. Native electrophoresis permits the separation of ETC complexes into their active forms, and these complexes can then be studied using immunoblotting, in-gel assays (IGA), and purification by electroelution. By combining the results of native gel PAGE with those of other mitochondrial assays, it is possible to obtain a completer picture of ETC activity, its dynamic assembly and disassembly, and how this regulates mitochondrial structure and function. This work will also discuss limitations of these techniques. In summary, the technique of native PAGE, followed by immunoblotting, IGA, and electroelution, presented below, is a powerful way to investigate the functionality and composition of mitochondrial ETC supercomplexes.

This protocol describes the separation of functional mitochondrial electron transport chain complexes (Cx) I-V and supercomplexes thereof using native electrophoresis to reveal information about their assembly and structure. The native gel can be subjected to immunoblotting, in-gel assays, and purification by electroelution to further characterize individual complexes.

The mitochondrial electron transport chain (ETC) transduces the energy derived from the breakdown of various fuels into the bioenergetic currency of the ...

Semi-automated Biopanning of Bacterial Display Libraries for Peptide Affinity Reagent Discovery and Analysis of Resulting Isolates

Biopanning bacterial display libraries is a proven technique for peptide affinity reagent discovery for recognition of both biotic and abiotic targets. Peptide affinity reagents can be used for similar applications to antibodies, including sensing and therapeutics, but are more robust and able to perform in more extreme environments. Specific enrichment of peptide capture agents to a protein target of interest is enhanced using semi-automated sorting methods which improve binding and wash steps and therefore decrease the occurrence of false positive binders. A semi-automated sorting method is described herein for use with a commercial automated magnetic-activated cell sorting device with an unconstrained bacterial display sorting library expressing random 15-mer peptides. With slight modifications, these methods are extendable to other automated devices, other sorting libraries, and other organisms. A primary goal of this work is to provide a comprehensive methodology and expound the thought process applied in analyzing and minimizing the resulting pool of candidates. These techniques include analysis of on-cell binding using fluorescence-activated cell sorting (FACS), to assess affinity and specificity during sorting and in comparing individual candidates, and the analysis of peptide sequences to identify trends and consensus sequences for understanding and potentially improving the affinity to and specificity for the target of interest.

Biopanning bacterial display libraries is a proven technique for discovery of peptide affinity reagents, a robust alternative to antibodies. The semi-automated sorting method herein has streamlined biopanning to decrease the occurrence of false positives. Here we illustrate the thought process and techniques applied in evaluating candidates and minimizing downstream analysis.

Biopanning bacterial display libraries is a proven technique for peptide affinity reagent discovery for recognition of both biotic and abiotic targets. ...

A Simple Method for High Throughput Chemical Screening in Caenorhabditis Elegans

Caenorhabditis elegans is a useful organism for testing chemical effects on physiology. Whole organism small molecule screens offer significant advantages for identifying biologically active chemical structures that can modify complex phenotypes such as lifespan. Described here is a simple protocol for producing hundreds of 96-well culture plates with fairly consistent numbers of C. elegans in each well. Next, we specified how to use these cultures to screen thousands of chemicals for effects on the lifespan of the nematode C. elegans. This protocol makes use of temperature sensitive sterile strains, agar plate conditions, and simple animal handling to facilitate the rapid and high throughput production of synchronized animal cultures for screening.

A Simple Method for High Throughput Chemical Screening in Caenorhabditis Elegans

Here we describe a simple protocol for rapidly producing hundreds of nematode growth media agar, 96-well culture plates with consistent numbers of Caenorhhabditis elegans per well. These cultures are useful for the phenotypic screening of whole organisms. We focus here on using these cultures to screen chemicals for pro-longevity effects.

Caenorhabditis elegans is a useful organism for testing chemical effects on physiology. Whole organism small molecule screens offer significant advantages ...

Measuring Trans-Plasma Membrane Electron Transport by C2C12 Myotubes

Trans-plasma membrane electron transport (tPMET) plays a role in protection of cells from intracellular reductive stress as well as protection from damage by extracellular oxidants. This process of transporting electrons from intracellular reductants to extracellular oxidants is not well defined. Here we present spectrophotometric assays by C2C12 myotubes to monitor tPMET utilizing the extracellular electron acceptors: water-soluble tetrazolium salt-1 (WST-1) and 2,6-dichlorophenolindophenol (DPIP or DCIP). Through reduction of these electron acceptors, we are able to monitor this process in a real-time analysis. With the addition of enzymes such as ascorbate oxidase (AO) and superoxide dismutase (SOD) to the assays, we can determine which portion of tPMET is due to ascorbate export or superoxide production, respectively. While WST-1 was shown to produce stable results with low background, DPIP was able to be re-oxidized after the addition of AO and SOD, which was demonstrated with spectrophotometric analysis. This method demonstrates a real-time, multi-well, quick spectrophotometric assay with advantages over other methods used to monitor tPMET, such as ferricyanide (FeCN) and ferricytochrome c reduction.

The goal of this protocol is to spectrophotometrically monitor trans-plasma membrane electron transport utilizing extracellular electron acceptors and to analyze enzymatic interactions that may occur with these extracellular electron acceptors.

Trans-plasma membrane electron transport (tPMET) plays a role in protection of cells from intracellular reductive stress as well as protection from damage ...

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.

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.

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

Extracellular Protein Microarray Technology for High Throughput Detection of Low Affinity Receptor-Ligand Interactions

Secreted factors, membrane-tethered receptors, and their interacting partners are main regulators of cellular communication and initiation of signaling cascades during homeostasis and disease, and as such represent prime therapeutic targets. Despite their relevance, these interaction networks remain significantly underrepresented in current databases; therefore, most extracellular proteins have no documented binding partner. This discrepancy is primarily due to the challenges associated with the study of the extracellular proteins, including expression of functional proteins, and the weak, low affinity, protein interactions often established between cell surface receptors. The purpose of this method is to describe the printing of a library of extracellular proteins in a microarray format for screening of protein-protein interactions. To enable detection of weak interactions, a method based on multimerization of the query protein under study is described. Coupled to this microbead-based multimerization approach for increased multivalency, the protein microarray allows robust detection of transient protein-protein interactions in high throughput. This method offers a rapid and low sample consuming-approach for identification of new interactions applicable to any extracellular protein. Protein microarray printing and screening protocol are described. This technology will be useful for investigators seeking a robust method for discovery of protein interactions in the extracellular space.

Here, we present a protocol to screen extracellular protein microarrays for identification of novel receptor-ligand interactions in high throughput. We also describe a method to enhance detection of transient protein-protein interactions by using protein-microbead complexes.

Secreted factors, membrane-tethered receptors, and their interacting partners are main regulators of cellular communication and initiation of signaling ...

Analysis of &#946;-Amyloid-induced Abnormalities on Fibrin Clot Structure by Spectroscopy and Scanning Electron Microscopy

This article presents methods for generating in vitro fibrin clots and analyzing the effect of beta-amyloid (A&#946;) protein on clot formation and structure by spectrometry and scanning electron microscopy (SEM). A&#946;, which forms neurotoxic amyloid aggregates in Alzheimer&#39;s disease (AD), has been shown to interact with fibrinogen. This A&#946;-fibrinogen interaction makes the fibrin clot structurally abnormal and resistant to fibrinolysis. A&#946;-induced abnormalities in fibrin clotting may also contribute to cerebrovascular aspects of the AD pathology such as microinfarcts, inflammation, as well as, cerebral amyloid angiopathy (CAA). Given the potentially critical role of neurovascular deficits in AD pathology, developing compounds which can inhibit or lessen the A&#946;-fibrinogen interaction has promising therapeutic value. In vitro methods by which fibrin clot formation can be easily and systematically assessed are potentially useful tools for developing therapeutic compounds. Presented here is an optimized protocol for in vitro generation of the fibrin clot, as well as analysis of the effect of A&#946; and A&#946;-fibrinogen interaction inhibitors. The clot turbidity assay is rapid, highly reproducible and can be used to test multiple conditions simultaneously, allowing for the screening of large numbers of A&#946;-fibrinogen inhibitors. Hit compounds from this screening can be further evaluated for their ability to ameliorate A&#946;-induced structural abnormalities of the fibrin clot architecture using SEM. The effectiveness of these optimized protocols is demonstrated here using TDI-2760, a recently identified A&#946;-fibrinogen interaction inhibitor.

Presented here are two methods that can be used individually or in combination to analyze the effect of beta-amyloid on fibrin clot structure. Included is a protocol for creating an in vitro fibrin clot, followed by clot turbidity and scanning electron microscopy methods.

This article presents methods for generating in vitro fibrin clots and analyzing the effect of beta-amyloid (A&#946;) protein on clot formation and ...