Funding provided by Slippery Rock University. We would like to thank T. Boron III (Slippery Rock University) and C. Haney (University of Pennsylvania) for their insights.

1. Hydrolysis of Ni-Schiff-Base Complex
<ol>
	<li>Dissolve 1 mmol of the Ni-PBP-Schiff-base complex in 40 mL of N,N-dimethylformamide (DMF) with stirring in a 250 mL round bottom flask at room temperature.</li>
	<li>Add 60 mL of 0.2 M aqueous EDTA solution, pH 4.5.</li>
	<li>Using a magnetic stir bar and stir plate, stir the combined solution overnight. As the Schiff-base complex is hydrolyzed, the color will shift from a deep red to white.</li>
	<li>After completion of the reaction as indicated by the absence of any red coloring, transfer the reaction to a 250 mL separatory funnel.</li>
	<li>Add 50 mL dichloromethane, cap the separatory funnel, and mix. Drain the organic wash into a waste beaker. Repeat this process three times to remove PBP and any residual Ni-PBP-Schiff-base complex. Collect the remaining aqueous layer in a 250 mL round bottom flask.</li>
</ol>
2. Fmoc Protection of Hydrolyzed Amino Acid
<ol>
	<li>Adjust the isolated aqueous layer from step 1.5 described above to pH 7 using solid sodium bicarbonate. Check pH periodically using pH test strips.</li>
	<li>Add 168 mg sodium bicarbonate (2.00 mmol, 2 equiv) to the solution and stir using a magnetic stir bar and stir plate.</li>
	<li>Dissolve 337 mg Fmoc N-hydroxysuccinimide ester (1.00 mmol, 1 equiv) in a minimal amount of dioxane (roughly 4 or 5 mL) in a 10 mL vial. Transfer this solution to the aqueous solution and stir overnight.</li>
	<li>After allowing the reaction to stir overnight, acidify the resulting solution to pH 2 with 1 M hydrochloric acid. Check pH periodically using pH test strips.</li>
	<li>Transfer the reaction to a 250 mL separatory funnel and add 50 mL ethyl acetate. Cap the separatory funnel, mix well, and collect the organic layer in a 250 mL Erlenmeyer flask. Repeat this process two additional times, combining the organic extracts. Dry the combined organic extracts with roughly 3 g of magnesium sulfate.</li>
	<li>Concentrate the combined organic extracts using a rotary evaporator to afford the crude Fmoc-protected amino acid.</li>
</ol>

<ol>
	<li>Wang, J., Shi, T., Deng, G., Jiang, H., Liu, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Highly+Enantio-+and+Diastereoselective+Mannich+Reactions+of+Chiral+Ni(II)+Glycinates+with+amino+sulfones.+Efficient+asymmetric+synthesis+of+aromatic+&#945;,&#946;-diamino+acids.">Highly Enantio- and Diastereoselective Mannich Reactions of Chiral Ni(II) Glycinates with amino sulfones. Efficient asymmetric synthesis of aromatic &#945;,&#946;-diamino acids.</a> J. Org. Chem. 73 (21), 8563-8570 (2011).</li><li>Wang, J., Lin, D., Zhou, S., Ding, X., Soloshonok, V. A., Liu, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Asymmetric+synthesis+of+sterically+and+electronically+demanding+linear+&#969;,-trifluoromethyl+containing+amino+acids+via+alkylation+of+chiral+equivalents+of+nucleophilic+glycine+and+alanine.">Asymmetric synthesis of sterically and electronically demanding linear &#969;,-trifluoromethyl containing amino acids via alkylation of chiral equivalents of nucleophilic glycine and alanine.</a> J. Org. Chem. 76 (2), 684-687 (2011).</li><li>Wang, J., Zhou, S., Lin, D., Ding, X., Jiang, H., Liu, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Highly+diastereo-+and+enantioselective+synthesis+of+syn-&#946;,-substituted+tryptophans+via+asymmetric+Michael+addition+of+a+chiral+equivalent+of+nucleophilic+glycine+and+sulfonylindoles.">Highly diastereo- and enantioselective synthesis of syn-&#946;,-substituted tryptophans via asymmetric Michael addition of a chiral equivalent of nucleophilic glycine and sulfonylindoles.</a> Chem. Commun. 47 (29), 8355-8357 (2011).</li><li>Belokon, Y. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Highly+efficient+catalytic+synthesis+of+&#945;,-amino+acids+under+phase-transfer+conditions+with+a+novel+catalyst/substrate+pair.">Highly efficient catalytic synthesis of &#945;,-amino acids under phase-transfer conditions with a novel catalyst/substrate pair.</a> Angew. Chem. Int. Ed. 40 (10), 1948-1951 (2001).</li><li>Zhou, S., Wang, J., Lin, D., Zhao, F., Liu, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Enantioselective+synthesis+of+2-substituted-tetrahydroisoquinolin-1-yl+glycine+derivatives+via+oxidative+cross-dehydrogenative+coupling+of+tertiary+amines+and+chiral+nickel(II)+glycinate.">Enantioselective synthesis of 2-substituted-tetrahydroisoquinolin-1-yl glycine derivatives via oxidative cross-dehydrogenative coupling of tertiary amines and chiral nickel(II) glycinate.</a> J. Org. Chem. 78 (22), 11204-11212 (2013).</li><li>Belokon, Y. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Synthesis+of+&#945;,-amino+acids+via+asymmetric+phase+transfer-catalyzed+alkylation+of+achiral+nickel(II)+complexes+of+glycine-derived+Schiff+bases.">Synthesis of &#945;,-amino acids via asymmetric phase transfer-catalyzed alkylation of achiral nickel(II) complexes of glycine-derived Schiff bases.</a> J. Am. Chem. Soc. 125 (42), 12860-12871 (2003).</li><li>Ueki, H., Ellis, T. K., Martin, C. H., Soloshonok, V. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Efficient+large-scale+synthesis+of+picolinic+acid-derived+nickel(II)+complexes+of+glycine.">Efficient large-scale synthesis of picolinic acid-derived nickel(II) complexes of glycine.</a> Eur. J. Org. Chem. 2003 (10), 1954-1957 (2003).</li><li>Dener, J. M., Fantauzzi, P. P., Kshirsagar, T. A., Kelly, D. E., Wolfe, A. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Large-scale+syntheses+of+Fmoc-protected+non-proteogenic+amino+acids:+useful+building+blocks+for+combinatorial+libraries.">Large-scale syntheses of Fmoc-protected non-proteogenic amino acids: useful building blocks for combinatorial libraries.</a> Org. Process Res. Dev. 5 (4), 445-449 (2001).</li><li>Cruz, L. J., Beteta, N. G., Ewenson, A., Albericio, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=&amp;#34;One-pot&amp;#34;,+preparation+of+N-carbamate+protected+amino+acids+via+the+azide.">&amp;#34;One-pot&amp;#34;, preparation of N-carbamate protected amino acids via the azide.</a> Org Process Res. Dev. 8 (6), 920-924 (2004).</li><li>Hart, J. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Ullmann's Encyclopedia of Industrial Chemistry. , (2000).</li><li>Adamson, J. G., Blaskovich, M. A., Groenevelt, H., Lajoie, G. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Simple+and+convenient+synthesis+of+tert-butyl+ethers+of+Fmoc-serine,+Fmoc-threonine,+and+Fmoc-tyrosine.">Simple and convenient synthesis of tert-butyl ethers of Fmoc-serine, Fmoc-threonine, and Fmoc-tyrosine.</a> J. Org. Chem. 56 (10), 3447-3449 (1991).</li><li>Seyfried, M. S., Lauber, B. S., Luedtke, N. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Multiple-turnover+isotopic+labeling+of+Fmoc-+and+Boc-protected+amino+acids+with+oxygen+isotopes.">Multiple-turnover isotopic labeling of Fmoc- and Boc-protected amino acids with oxygen isotopes.</a> Org. Lett. 12 (1), 104-106 (2010).</li><li>Bonke, G., Vedel, L., Witt, M., Jaroszewski, J. W., Olsen, C. A., Franzyk, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dimeric+building+blocks+for+solid-phase+synthesis+of+&#945;,-peptide-&#946;,-peptoid+chimeras.">Dimeric building blocks for solid-phase synthesis of &#945;,-peptide-&#946;,-peptoid chimeras.</a> Synthesis. 2008 (15), 2381-2390 (2008).</li></ol>

The authors have nothing to disclose.

The protocol described above is useful in its ability to facilitate the isolation of an amino acid backbone from a Ni-Schiff-base complex under mild pH conditions and subsequent Fmoc protection of this isolated amino acid through two critical steps. The first step involves stirring a DMF/water solution containing EDTA to facilitate release of the amino acid from the complex. Residual complex or organic byproducts can easily be removed with extraction. The second step of this protocol involves an Fmoc protection of the am...

Unnatural amino acids (UAA&#39;s) bearing side chains that vary from those of the twenty naturally occurring amino acids found in nature have found utility in a wide-range of applications. Synthesis of these UAA&#39;s, however, can be difficult depending on the structure of the side-chains and the stereochemistry of the amino acid backbone. C-H bond activation of glycine in the context of a nickel Schiff-base complex has been used to produce a variety of amino acid derivatives including &#945;,&#946;-diamino acids1 and UAA&#39;s bearing fluorinated2 or heterocyclic side-chains.3
After addition of unnatural side-chains, functionalized UAA&#39;s are typically removed from the Schiff-base complex by reflux in hydrochloric acid4 and are subsequently isolated using ion-exchange chromatography. While generally efficient, this protocol generates amino acids that may be unsuitable for use in solid-phase peptide synthesis (SPPS). The nature of SPPS requires the presence of acid-labile side-chain protecting groups and the strongly acidic nature of typical Ni-Schiff-base decomposition conditions prevents isolation of UAA&#39;s with these protecting groups intact. To our knowledge, only one alternative decomposition method has been reported: use of ethylenediaminetetraacetic acid (EDTA) and hydrazine at elevated temperatures,5 conditions that themselves may not be suitable for some side-chain protecting groups such as phthalimides.
<img alt="Figure 1" src="/files/ftp_upload/55677/55677fig1.jpg" /> 
Figure 1: Synthesis of Ni-PBP-Gly from Ni2+, PBP, and Glycine (Gly). <a href="http://cloudflare.jove.com/files/ftp_upload/55677/55677fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
Herein, we report a method for hydrolysis of a Ni-Schiff-base complex, Ni-PBP-Gly (Figure 1). This complex, derived from Ni2+, glycine, and pyridine-2-carboxylic acid(2-benzoyl-phenyl)-amide (PBP),6 has been demonstrated to be a useful platform for synthesis of a variety of UAA&#39;s and is easily accessible using a two-step synthetic route.7 Synthesis of this complex is literature-precedented in high yield.6 Our results described below demonstrate the applicability of hydrolysis conditions utilizing EDTA at mildly acidic to neutral pH conditions appropriate for use with UAA&#39;s bearing acid-labile side-chain protecting groups. Following hydrolysis, the resulting aqueous solution can be isolated and subjected immediately to standard Fmoc protection conditions to afford an Fmoc-protected amino acid (Figure 2).
<img alt="Figure 2" src="/files/ftp_upload/55677/55677fig2.jpg" /> 
Figure 2: Hydrolysis and Fmoc-protection of an Amino Acid Isolated from Ni-PBP-Gly. Reaction Conditions: i. EDTA (12 equiv), pH 4.5; ii. Ethyl acetate wash and adjustment to pH 7; iii. Fmoc-OSu (1 equiv), NaHCO3 (2 equiv). <a href="http://cloudflare.jove.com/files/ftp_upload/55677/55677fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Ni-PBP-Gly</td>
			<td></td>
			<td></td>
			<td>Synthesized from published protocol</td>
		</tr>
		<tr>
			<td>DMF</td>
			<td>Fisher</td>
			<td>D119-4</td>
			<td></td>
		</tr>
		<tr>
			<td>EDTA</td>
			<td>Fisher</td>
			<td>S311-100</td>
			<td></td>
		</tr>
		<tr>
			<td>Dichloromethane</td>
			<td>Acros</td>
			<td>AC610050040</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium Bicarbonate</td>
			<td>Fisher</td>
			<td>S233-500</td>
			<td></td>
		</tr>
		<tr>
			<td>Fmoc-OSu</td>
			<td>Chem-Impex</td>
			<td>&#34;00147&#34;</td>
			<td></td>
		</tr>
		<tr>
			<td>Dioxane</td>
			<td>Fisher</td>
			<td>D111-500</td>
			<td></td>
		</tr>
		<tr>
			<td>Hydrochloric Acid</td>
			<td>Fisher</td>
			<td>A144-500</td>
			<td></td>
		</tr>
		<tr>
			<td>Ethyl Acetate</td>
			<td>Acros</td>
			<td>AC610060040</td>
			<td></td>
		</tr>
		<tr>
			<td>Magnesium Sulfate</td>
			<td>Fisher</td>
			<td>M65-500</td>
			<td></td>
		</tr>
		<tr>
			<td>ZEOPrep 60ECO Silica Gel</td>
			<td>ZEOChem</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Hexanes</td>
			<td>Fisher</td>
			<td>3200250.650.443</td>
			<td></td>
		</tr>
		<tr>
			<td>Chromatography Column</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>pH Test Strips</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Rotary Evaporator</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>250 mL Separatory Funnel</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>250 mL Round Bottom Flask</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Stir Bar</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Stir Plate</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
	</tbody>
</table>

hydrolysis of a ni-schiff-base complex using conditions suitable for retention of acid-labile protecting groups

Unnatural amino acids, amino acids containing side-chain functionalities not commonly seen in nature, are increasingly found in synthetic peptide sequences. Synthesis of some unnatural amino acids often includes the use of a precursor consisting of a Schiff-base stabilized by a nickel cation. Unnatural side-chains can be installed on an amino acid backbone found in this Schiff-base complex. The resulting unnatural amino acid can then be isolated from this complex using hydrolysis of the Schiff-base, typically by employing reflux in strongly acidic solution. These highly acidic conditions may remove acid-labile side-chain protecting groups necessary for the unnatural amino acids to be used in microwave-assisted solid-phase peptide synthesis. In this work, we present an efficient hydrolysis and subsequent Fmoc protection of an amino acid isolated from a Ni-Schiff base complex. Hydrolysis conditions presented in this work are suitable for retention of acid-labile side-chain protecting groups and may be adaptable to a variety of unnatural amino acid substrates.

We hypothesized that removal of the Ni2+ from the Ni-PBP-Gly complex could allow an efficient aqueous hydrolysis of the Schiff-base without the need for harsh pH conditions. As EDTA is an inexpensive and well-studied chelating agent,10 we hypothesized that addition of EDTA to a solution of Ni-PBP-Gly would facilitate chelation of Ni2+ ions, thereby promoting hydrolysis of the complex.
<p class="jove_content" fo:keep-together.within-page="1...

Watch this Scientific Journal Video about Hydrolysis of a Ni-Schiff-Base Complex Using Conditions Suitable for Retention of Acid-labile Protecting Groups at JoVE.com

Hydrolysis of a Ni-Schiff-Base Complex Using Conditions Suitable for Retention of Acid-labile Protecting Groups

Here, we present an efficient hydrolysis and subsequent Fmoc protection of an amino acid isolated from a Ni-Schiff-base complex. Hydrolysis conditions presented here are suitable for use when retention of acid-labile side-chain protecting groups is required. This technique may be adaptable to a variety of unnatural amino acid substrates.

Unnatural amino acids, amino acids containing side-chain functionalities not commonly seen in nature, are increasingly found in synthetic peptide ...

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Biochemistry

9.6K Views.  Slippery Rock University. Here, we present an efficient hydrolysis and subsequent Fmoc protection of an amino acid isolated from a Ni-Schiff-base complex. Hydrolysis conditions presented here are suitable for use when retention of acid-labile side-chain protecting groups is required. This technique may be adaptable to a variety of unnatural amino acid substrates.

Video: Hydrolysis of a Ni-Schiff-Base Complex Using Conditions Suitable for Retention of Acid-labile Protecting Groups

Protein Complex Affinity Capture from Cryomilled Mammalian Cells

Affinity capture is an effective technique for isolating endogenous protein complexes for further study. When used in conjunction with an antibody, this technique is also frequently referred to as immunoprecipitation. Affinity capture can be applied in a bench-scale and in a high-throughput context. When coupled with protein mass spectrometry, affinity capture has proven to be a workhorse of interactome analysis. Although there are potentially many ways to execute the numerous steps involved, the following protocols implement our favored methods. Two features are distinctive: the use of cryomilled cell powder to produce cell extracts, and antibody-coupled paramagnetic beads as the affinity medium. In many cases, we have obtained superior results to those obtained with more conventional affinity capture practices. Cryomilling avoids numerous problems associated with other forms of cell breakage. It provides efficient breakage of the material, while avoiding denaturation issues associated with heating or foaming. It retains the native protein concentration up to the point of extraction, mitigating macromolecular dissociation. It reduces the time extracted proteins spend in solution, limiting deleterious enzymatic activities, and it may reduce the non-specific adsorption of proteins by the affinity medium. Micron-scale magnetic affinity media have become more commonplace over the last several years, increasingly replacing the traditional agarose- and Sepharose-based media. Primary benefits of magnetic media include typically lower non-specific protein adsorption; no size exclusion limit because protein complex binding occurs on the bead surface rather than within pores; and ease of manipulation and handling using magnets.

Here we describe protocols to disrupt mammalian cells by solid-state milling at a cryogenic temperature, produce a cell extract from the resulting cell powder, and isolate protein complexes of interest by affinity capture upon antibody-coupled micron-scale paramagnetic beads.

Affinity capture is an effective technique for isolating endogenous protein complexes for further study. When used in conjunction with an antibody, this ...

A Method for Measuring Metabolism in Sorted Subpopulations of Complex Cell Communities Using Stable Isotope Tracing

Mammalian cell types exhibit specialized metabolism, and there is ample evidence that various co-existing cell types engage in metabolic cooperation. Moreover, even cultures of a single cell type may contain cells in distinct metabolic states, such as resting or cycling cells. Methods for measuring metabolic activities of such subpopulations are valuable tools for understanding cellular metabolism. Complex cell populations are most commonly separated using a cell sorter, and subpopulations isolated by this method can be analyzed by metabolomics methods. However, a problem with this approach is that the cell sorting procedure subjects cells to stresses that may distort their metabolism.
To overcome these issues, we reasoned that the mass isotopomer distributions (MIDs) of metabolites from cells cultured with stable isotope-labeled nutrients are likely to be more stable than absolute metabolite concentrations, because MIDs are formed over longer time scales and should be less affected by short-term exposure to cell sorting conditions. Here, we describe a method based on this principle, combining cell sorting with liquid chromatography-high resolution mass spectrometry (LC-HRMS). The procedure involves analyzing three types of samples: (1) metabolite extracts obtained directly from the complex population; (2) extracts of "mock sorted" cells passed through the cell sorter instrument without gating any specific population; and (3) extracts of the actual sorted populations. The mock sorted cells are compared against direct extraction to verify that MIDs are indeed not altered by the cell sorting procedure itself, prior to analyzing the actual sorted populations. We show example results from HeLa cells sorted according to cell cycle phase, revealing changes in nucleotide metabolism.

This article describes a method for studying cellular metabolism in complex communities of multiple cell types, using a combination of stable isotope tracing, cell sorting to isolate specific cell types, and mass spectrometry.

Mammalian cell types exhibit specialized metabolism, and there is ample evidence that various co-existing cell types engage in metabolic cooperation. ...

Preparation of Giant Vesicles Encapsulating Microspheres by Centrifugation of a Water-in-oil Emulsion

The constructive biology and the synthetic biology approach to creating artificial life involve the bottom-up assembly of biological or nonbiological materials. Such approaches have received considerable attention in research on the boundary between living and nonliving matter and have been used to construct artificial cells over the past two decades. In particular, Giant Vesicles (GVs) have often been used as artificial cell membranes. In this paper, we describe the preparation of GVs encapsulating highly packed microspheres as a model of cells containing highly condensed biomolecules. The GVs were prepared by means of a simple water-in-oil emulsion centrifugation method. Specifically, a homogenizer was used to emulsify an aqueous solution containing the materials to be encapsulated and an oil containing dissolved phospholipids, and the resulting emulsion was layered carefully on the surface of another aqueous solution. The layered system was then centrifuged to generate the GVs. This powerful method was used to encapsulate materials ranging from small molecules to microspheres.

Giant vesicles containing highly packed micrometer-sized components are useful cell models. The water-in-oil emulsion centrifugation method is a simple, powerful tool for the preparation of giant vesicles with encapsulated materials.

The constructive biology and the synthetic biology approach to creating artificial life involve the bottom-up assembly of biological or nonbiological ...

A Modified Yeast-one Hybrid System for Heteromeric Protein Complex-DNA Interaction Studies

Over the years, the yeast one-hybrid assay has proven to be an important technique for the identification and validation of physical interactions between proteins such as transcription factors (TFs) and their DNA target. The method presented here utilizes the underlying concept of the Y1H but is modified further to study and validate protein complexes binding to their target DNA. Hence, it is referred to as the modified yeast one-hybrid (Y1.5H) assay. This assay is cost effective and can be easily performed in a regular laboratory setting. Albeit using a heterologous system, the described method could be a valuable tool to test and validate the heteromeric protein complex binding to their DNA target(s) for functional genomics in any system of study, especially plant genomics.

The modified yeast one-hybrid assay described here is an extension of the classical yeast one-hybrid (Y1H) assay to study and validate the heteromeric protein complex-DNA interaction in a heterologous system for any functional genomics study.

Over the years, the yeast one-hybrid assay has proven to be an important technique for the identification and validation of physical interactions between ...

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.

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

Toeprinting Analysis of Translation Initiation Complex Formation on Mammalian mRNAs

Translation initiation is the rate-limiting step of protein synthesis and represents a key point at which cells regulate their protein output. Regulation of protein synthesis is the key to cellular stress-response, and dysregulation is central to many disease states, such as cancer. For instance, although cellular stress leads to the inhibition of global translation by attenuating cap-dependent initiation, certain stress-response proteins are selectively translated in a cap-independent manner. Discreet RNA regulatory elements, such as cellular internal ribosome entry sites (IRESes), allow for the translation of these specific mRNAs. Identification of such mRNAs, and the characterization of their regulatory mechanisms, have been a key area in molecular biology. Toeprinting is a method for the study of RNA structure and function as it pertains to translation initiation. The goal of toeprinting is to assess the ability of in vitro transcribed RNA to form stable complexes with ribosomes under a variety of conditions, in order to determine which sequences, structural elements, or accessory factors are involved in ribosome binding&#8212;a pre-cursor for efficient translation initiation. Alongside other techniques, such as western analysis and polysome profiling, toeprinting allows for a robust characterization of mechanisms for the regulation of translation initiation.

Toeprinting aims to measure the ability of in vitro transcribed RNA to form translation initiation complexes with ribosomes under a variety of conditions. This protocol describes a method for toeprinting mammalian RNA and can be used to study both cap-dependent and IRES-driven translation.

Translation initiation is the rate-limiting step of protein synthesis and represents a key point at which cells regulate their protein output. Regulation ...

Medium Preparation for the Cultivation of Microorganisms under Strictly Anaerobic/Anoxic Conditions

In contrast to aerobic organisms, strictly anaerobic microorganisms require the absence of oxygen and usually a low redox potential to initiate growth. As oxygen is ubiquitous in air, retaining O2-free conditions during all steps of cultivation is challenging but a prerequisite for anaerobic culturing. The protocol presented here demonstrates the successful cultivation of an anaerobic mixed culture derived from a biogas plant using a simple and inexpensive method. A precise description of the entire anoxic culturing process is given including media preparation, filling of cultivation flasks, supplementation with redox indicator and reducing agents to provide low redox potentials as well as exchanging the headspace to keep media free from oxygen. Furthermore, a detailed overview of aseptically inoculating gas tight serum flasks (by using sterile syringes and needles) and suitable incubation conditions is provided. The present protocol further deals with gas and liquid sampling for subsequent analyses regarding gas composition and volatile fatty acid concentrations using gas chromatography (GC) and high performance liquid chromatography (HPLC), respectively, and the calculation of biogas and methane yield considering the ideal gas law.

As obligate anaerobic organisms are unable to grow upon oxygen exposure, the use of anaerobic culturing techniques is indispensable. Here, we demonstrate a simple and effective method to cultivate a mixed culture derived from a biogas plant from media preparation to gas and volatile fatty acid quantification.

In contrast to aerobic organisms, strictly anaerobic microorganisms require the absence of oxygen and usually a low redox potential to initiate growth. As ...

Crystallization and Structural Determination of an Enzyme:Substrate Complex by Serial Crystallography in a Versatile Microfluidic Chip

The preparation of well diffracting crystals and their handling before their X-ray analysis are two critical steps of biocrystallographic studies. We describe a versatile microfluidic chip that enables the production of crystals by the efficient method of counter-diffusion. The convection-free environment provided by the microfluidic channels is ideal for crystal growth and useful to diffuse a substrate into the active site of the crystalline enzyme. Here we applied this approach to the CCA-adding enzyme of the psychrophilic bacterium Planococcus halocryophilus in the presented example. After crystallization and substrate diffusion/soaking, the crystal structure of the enzyme:substrate complex was determined at room temperature by serial crystallography and the analysis of multiple crystals directly inside the chip. The whole procedure preserves the genuine diffraction properties of the samples because it requires no crystal handling.

A versatile microfluidic device is described that enables the crystallization of an enzyme using the counter-diffusion method, the introduction of a substrate in the crystals by soaking, and the 3D structure determination of the enzyme:substrate complex by a serial analysis of crystals inside the chip at room temperature.

The preparation of well diffracting crystals and their handling before their X-ray analysis are two critical steps of biocrystallographic studies. We ...

Complementation of Splicing Activity by a Galectin-3 - U1 snRNP Complex on Beads

Classic depletion-reconstitution experiments indicate that galectin-3 is a required splicing factor in nuclear extracts. The mechanism of incorporation of galectin-3 into the splicing pathway is addressed in this paper. Sedimentation of HeLa cell nuclear extracts on 12%-32% glycerol gradients yields fractions enriched in an endogenous ~10S particle that contains galectin-3 and U1 snRNP. We now describe a protocol to deplete nuclear extracts of U1 snRNP with concomitant loss of splicing activity. Splicing activity in the U1-depleted extract can be reconstituted by the galectin-3 - U1 snRNP particle trapped on agarose beads covalently coupled with anti-galectin-3 antibodies. The results indicate that the galectin-3 - U1 snRNP - pre-mRNA ternary complex is a functional E complex leading to intermediates and products of the splicing reaction and that galectin-3 enters the splicing pathway through its association with U1 snRNP. The scheme of using complexes affinity- or immuno-selected on beads to reconstitute splicing activity in extracts depleted of a specific splicing factor may be generally applicable to other systems.

This article describes the experimental procedures for (a) depletion of U1 snRNP from nuclear extracts, with concomitant loss of splicing activity; and (b) reconstitution of splicing activity in the U1-depleted extract by galectin-3 - U1 snRNP particles bound to beads covalently coupled with anti-galectin-3 antibodies.

Classic depletion-reconstitution experiments indicate that galectin-3 is a required splicing factor in nuclear extracts. The mechanism of incorporation of ...

RIBO-seq in Bacteria: a Sample Collection and Library Preparation Protocol for NGS Sequencing

The ribosome profiling technique (RIBO-seq) is currently the most effective tool for studying the process of protein synthesis in vivo. The advantage of this method, in comparison to other approaches, is its ability to monitor translation by precisely mapping the position and number of ribosomes on a mRNA transcript.
In this article, we describe the consecutive stages of sample collection and preparation for RIBO-seq method in bacteria, highlighting the details relevant to the planning and execution of the experiment.
Since the RIBO-seq relies on intact ribosomes and related mRNAs, the key step is rapid inhibition of translation and adequate disintegration of cells. Thus, we suggest filtration and flash-freezing in liquid nitrogen for cell harvesting with an optional pretreatment with chloramphenicol to arrest translation in bacteria. For the disintegration, we propose grinding frozen cells with mortar and pestle in the presence of aluminum oxide to mechanically disrupt the cell wall. In this protocol, sucrose cushion or a sucrose gradient ultracentrifugation for monosome purification is not required. Instead, mRNA separation using polyacrylamide gel electrophoresis (PAGE) followed by the ribosomal footprint excision (28-30 nt band) is applied and provides satisfactory results. This largely simplifies the method as well as reduces the time and equipment requirements for the procedure. For library preparation, we recommend using the commercially available small RNA kit for Illumina sequencing from New England Biolabs, following manufacturer&#39;s guidelines with some degree of optimization.
The resulting cDNA libraries present appropriate quantity and quality required for next generation sequencing (NGS). Sequencing of the libraries prepared according to the described protocol results in 2 to 10 mln uniquely mapped reads per sample providing sufficient data for comprehensive bioinformatic analysis. The protocol we present is quick and relatively easy and can be performed with standard laboratory equipment.

Here we describe the stages of sample collection and preparation for RIBO-seq in bacteria. Sequencing of the libraries prepared according to these guidelines results in sufficient data for comprehensive bioinformatic analysis. The protocol we present is simple, uses standard laboratory equipment and takes seven days from lysis to obtaining libraries.

The ribosome profiling technique (RIBO-seq) is currently the most effective tool for studying the process of protein synthesis in vivo. The advantage of ...