This research was supported by Award Number R00RR024105 from the National Center For Research Resources, National Institute of Health.

1. Reductive Methylation of Lysozyme
<ol>
	<li>Prepare 500 ml of a 50 mM sodium phosphate buffer at pH 7.5. Store at room temperature.</li>
	<li>Label three 1 ml microcentrifuge tubes for fully labeled sample (FLS), low pH partially labeled sample (LpH), and high pH partially labeled sample (HpH).</li>
	<li>Weigh out 10 mg of lysozyme and dissolve in 2 ml of sodium phosphate buffer to create an aqueous solution with a concentration of 5 mg/ml.&#160;Cover the tubes with aluminum foil to protect the formaldehyde from degradation due light exposure. Add 500 &#956;l of lysozyme solution to each tube.</li>
	<li>Prepare fresh 1.0 M solutions of dimethylamine borane complex (DMAB) and 13C-formaldehyde. Store at 0 &#176;C.</li>
	<li>For the fully labeled sample, use a molar ratio of 1:10 (reactive amine: formaldehyde), and for the partially labeled samples, use a 1:5 ratio. Alternatively, the ratio can be optimized. For example, in order to see the dominance of the &#945;-13C-dimethylamine peak at low pH, a 2:7 ratio was required.</li>
	<li>Add 6.1 &#956;l of 1.0 M DMAB to FLS, add 3.2 &#956;l of 1.0 M DMAB to LpH and HpH, and gently vortex. Then add 12.3 &#956;l of 1.0 M 13C-formaldehyde to FLS and 6.1 &#956;l of 1.0 M 13C-formaldehyde to LpH and HpH. Shake the reaction mixtures at 4 &#176;C for 2 hr.</li>
	<li>Add another aliquot of 1.0 M DMAB and 13C-formaldehyde as in the previous step and shake the reaction mixture overnight at 4 &#176;C for a total reaction time of 18-24 hr.</li>
	<li>Exchange the sample&#39;s buffer to phosphate at pH 7.5 to remove excess reagents and side products using a centrifugal filter (3 kDa molecular weight cut-off).</li>
	<li>For the partially labeled samples, reductively methylate the samples at pH 7.5 with natural abundance formaldehyde to create a homogeneously modified protein. Prepare new 1.0 M solutions of DMAB and natural abundance formaldehyde.</li>
	<li>Cover the sample tubes with foil, add 6.1 &#956;l of DMAB to LpH and HpH, and gently vortex. Then add 12.3 &#956;l of natural abundance formaldehyde. Shake the reaction mixtures at 4 &#176;C for 2 hr.</li>
	<li>Add another aliquot of 1.0 M DMAB and natural abundance formaldehyde as in the previous step and shake the reaction mixtures overnight at 4 &#176;C for a total reaction time of 18-24 hr. Then buffer exchange using steps 2.5-2.6&#160;and store 50 &#956;g samples at -20 &#176;C for MS.</li>
</ol>
2. Prepare the Sample for NMR Analysis
<ol>
	<li>Prepare a 2.0 M sodium borate buffer solution at pH 8.5 in H2O.</li>
	<li>Add 375 &#956;l of 2.0 M sodium borate buffer solution to four 15 ml conical tubes. Parafilm the tops and poke holes. Place the flask in a lyophilizing flask and freeze the sample.</li>
	<li>Place the frozen sample on a lyophilizer to dry. Remove when dry.</li>
	<li>Using a pipette, add 15 ml of D2O to each tube and mix.</li>
	<li>Label three 4 ml centrifugal filters (3 kDa molecular weight cut-off) with FLS, LpH, and HpH. Add the corresponding sample to the filter tubes (~500 &#956;l). Add 3.0 ml of 50 mM sodium borate buffer at pH 8.5 to each tube. Make a balance tube for the third tube and concentrate the samples to 500 &#956;l using a centrifuge with a 35&#176; fixed angle rotor at 4 &#176;C and 7,500 rcf.</li>
	<li>Empty the trap under each filter. Add another 3.0 ml of buffer to the samples and concentrate the samples again to 500 &#956;l. Repeat step 2.5&#160;4x.</li>
	<li>Using the bicinchoninic acid (BCA) protein assay36, determine the concentration of each sample. Dilute the lysozyme samples with 50 mM sodium borate buffer to make 150 &#956;M samples.</li>
	<li>Transfer 530 &#956;l to a 5 mm NMR tube or 250 &#956;M to a 5 mm magnetic susceptibility matched tube.</li>
	<li>Prepare an 80 mM stock solution of 1,2-dichloroethane-13C2 in D2O. Store this stock solution at -80 &#176;C. Dilute to 24 mM in 80 &#956;l and transfer to coaxial insert tube to use as a reference in NMR analysis. Analyze the samples with NMR. Note: Run a 1D 1H-13C HSQC and integrate the peak areas for each resonance. Peak areas should be the same for fully labeled sample.</li>
</ol>
3. Aminopeptidase Degradation of Lysozyme
<ol>
	<li>Prepare a 5 mg/ml solution of lysozyme in ultrapure water.</li>
	<li>Add 250 &#956;l of the lysozyme solution to a clean, 1 ml centrifuge tube.</li>
	<li>Add 25 &#956;l of a 0.1 M tricine solution, pH 8.0, and 6 &#956;l of a 2.16 &#956;g/ml Aeromonas proteolytica aminopeptidase solution.</li>
	<li>Incubate at 37 &#176;C for 6 hr.</li>
	<li>Add an additional 6 &#956;l of the 2.16 &#956;g/ml aminopeptidase solution to the sample for a 1:50 molar ratio of aminopeptidase to lysozyme. Incubate for an addition 20 hr at 37 &#176;C.</li>
	<li>Desalt 30 &#956;l of the sample using a C18 spin column, (follow manufacturer&#39;s protocol). Store at -20 &#176;C for MALDI MS analysis.</li>
	<li>Prepare the remaining protein sample for NMR as described in protocol&#160;2).</li>
</ol>
4. Prepare Samples for Mass Spectrometry
<ol>
	<li>Using the 50 &#956;g of sample reserved in step 2.3, exchange the buffer to 100 mM ammonium bicarbonate, pH 8.0, and concentrate to approximately 50 &#956;l using a 1 ml centrifugal filter (3 kDa molecular weight cut-off). Transfer the sample to a 1 ml centrifuge tube and wrap in aluminum foil.</li>
	<li>Add 2.0 &#956;l of 500 mM dithiothreitol to reduce the disulfide bonds. Incubate the solution at 60 &#176;C for 1 hr.</li>
	<li>Add 2 &#956;l of freshly prepared 500 mM iodoacetamide to alkylate the thiol groups. Incubate at room temperature for 20 min.</li>
	<li>Remove the foil and add 1.0 &#956;l of the 500 mM dithiothreitol solution to quench the reaction.</li>
	<li>Add 2.0 &#956;g of sequencing grade trypsin to digest the protein. Incubate the mixture at 37 &#176;C for 16-24 hr.</li>
	<li>Add TFA until the pH is less than 2 to quench the digestion. (Formic and acetic acids can be used as well)</li>
	<li>Desalt the sample on a C18 spin column and elute the protein in 80% acetonitrile/0.1% TFA. Dry the sample using a vacuum concentrator.</li>
	<li>Reconstitute the sample in 5.0 &#956;l of 50% acetonitrile/0.1% TFA for MALDI MS analysis. Mix equal volumes of the reconstituted sample and a saturated solution of 2,5-dihydroxybenzoic acid matrix solution on the MALDI target plate. Analyze the samples on a MALDI MS. Note: Use tandem MS to confirm the identity of peptide peaks. Smooth the mass spectra and analyze. For each dimethylamine-containing peptide, determine the peak intensity and peak area of each isotope in the profile. Calculate the percentage of 13C-incorporation as described previously34.</li>
</ol>

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C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Immobilization+of+the+Aminopeptidase+from+Aeromonas+proteolytica+on+Mg2+/Al3++Layered+Double+Hydroxide+Particles.">Immobilization of the Aminopeptidase from Aeromonas proteolytica on Mg2+/Al3+ Layered Double Hydroxide Particles.</a> Acs Appl. Mater., &amp; Interfaces. 2 (10), 2828-2832 (2010).</li><li>Poncz, L., Dearborn, D. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+Resistance+to+Tryptic+Hydrolysis+of+Peptide-Bonds+Adjacent+to+N-epsilon,N-Dimethyllysyl+Residues.">The Resistance to Tryptic Hydrolysis of Peptide-Bonds Adjacent to N-epsilon,N-Dimethyllysyl Residues.</a> J. Biol. Chem. 258 (3), 1844-1850 (1983).</li></ol>

We have nothing to disclose.

Assigning the NMR signals of reductively 13C-methylated proteins is necessary to fully utilize this isotopic labeling method. The use of MS to aid in the assignment of the NMR signals thru correlation of the partial 13C-incorporation data is a promising technique. The advantage of this technique over other assignment methods is that only the primary amino acid sequence is needed. The MS-assisted assignment method is limited when the N-terminal amino acid is a lysine because the 13</sup...

Nuclear magnetic resonance (NMR) spectroscopy is a valuable structure elucidation tool for proteins1. NMR spectroscopy can be used to determine the solution structure of a protein in its native state. To overcome the low natural abundance of stable magnetic isotopes, it is necessary to incorporate 13C and 15N into the protein of interest. The most common method employed is recombinant expression in a bacterial host2-3. However, two disadvantages of bacterial host over-expression are it cannot produce post-translational modifications and does not work for all proteins3-4. When bacterial expression is not a viable route for protein production, over-expression in nonbacterial hosts can be used, but isotopic labeling is difficult and expensive5. Alternative expression methods for incorporating 13C and 15N isotopes into proteins for NMR analysis include sparse labeling techniques using metabolic precursors for methyl labeling6 and single 13C, 15N amino acids7-9. A chemical approach to sparse labeling used herein is the well-established reductive 13C-methylation reaction (Figure 1), where the primary amino groups on a protein - the N-terminal &#945;-amine and the lysine, side chain &#949;-amines - are methylated. Once monomethylamines are formed, the amine readily undergoes methylation again, due to the higher pKa value, to form dimethylamines.
Reductive methylation was first introduced as a method to chemically modify proteins by Means and Feeney10. The advantages of this reaction are its broad applicability and mild reactions conditions at buffered, physiological pH and low temperatures11,12.&#160;In the presence of formaldehyde and a reducing agent, such as dimethylamine borane complex (DMAB), the lysine &#949;-amino groups and the N-terminal &#945;-amino group are selectively methylated to produce dimethylated amines. Although formaldehyde is known to cross-link proteins through the formation of methylene bridges, this process is blocked by the reducing agent13,14.
Reductive methylation has been successfully used to study proteins with both NMR and x-ray crystallography. Reductive methylation is used to facilitate the crystallization of otherwise intractable proteins15. Hen egg white lysozyme was the first protein crystallized in its dimethylated form. The root-mean square difference between the heavy atoms in the methylated and unmethylated lysozyme structures is 0.40 &#197;16. This comparison demonstrates that the protein structure can be maintained after reductive methylation, making the reaction a viable, labeling tool for structure elucidation.
By using 13C-labeled formaldehyde in the reductive methylation reaction, 13C-dimethylated amines are produced. The 13C-dimethylamines are NMR-detectable probes that have been widely used to study protein dynamics, structure, and function. NMR and reductive 13C-methylation have been used to study protein-ligand and protein-protein interactions for the &#946;2 adrenergic receptor17, ribonuclease A18, lysozyme19, fd gene 5 protein20, and cytochrome c21. Similarly, structural and functional properties have been studied of reductively 13C-methylated ribonuclease A22, lysozyme23, fd gene 5 protein24, Clostridium pasteurianum ferredoxin25, Fc fragment of IgG26, apolipoprotein A-I27,&#160;and MIP-1&#945;28 The dynamics of the 13C-dimethylamino groups have been studied on concanavalin A29-30 and calmodulin31.
Even though reductive 13C-methylation has been used widely to study proteins with NMR, the labeling method has always been limited by the difficulties of assigning the NMR resonances26. Most assignment strategies for reductively 13C-methylated proteins have relied on small numbers of sites,20,28 known structural properties11,19,23,26,31,32. or extensive genetic modifications33. None of these studies successfully assigned all the 13C-dimethylamine peaks except for the calmodulin study, where the peaks were assigned by site-directed mutagenesis of each lysine33. In the study of MIP-1&#945; dimer formation, the use of mass spectrometry (MS) to aid in the NMR assignment of reductively 13C-methylated amines was first reported28. Matrix assisted laser desorption ionization time-of-flight (MALDI-TOF) MS was used to identify the lysine at the interface of the dimer. The partially methylated lysines of human MIP-1&#945; tryptic peptides were identified with MS and correlated with the appearance of mono- and dimethylamine signals of the intact protein observed in 2D 1H-13C HSQC NMR spectra28. Our group expanded on the use of MS and presented an assignment method that requires no prior knowledge of the protein&#39;s structure or properties other than the amino acid sequence34. This method is applicable to most proteins. One exception is when a protein has an N-terminal lysine because the MS isotopic profile of the N-terminal lysine &#945;- and &#949;-13C-dimethylamines cannot be independently measured.
Here we present two methods, one chemical and one enzymatic, to identify the N-terminal &#945;-dimethylamine and the side chain &#949;-dimethylamine sites of an N-terminal lysine residue. The first method was inspired by C&#243;rdova et al.&#160;who used pH to control the selectivity of protein acetylation35. The reaction favors the N-terminal &#945;-amine at low pH and the side chain &#949;-amines at high pH, allowing the protein &#945;- and &#949;-amino groups to be distinguished, in their studies, with capillary electrophoresis35. We demonstrate how high and low pH is used to alter the labeling of protein amino groups using the reductive 13C-methylation reaction and to allow application of the MS-assisted assignment strategy. The second method to assign the N-terminal &#945;- and &#949;-13C-dimethylamines takes advantage of the selective removal of the N-terminal residue(s) using recombinant Aeromonas proteolytica aminopeptidase.

<table border="1">
	<tbody>
		<tr>
			<td>Name of Material/ Equipment</td>
			<td>Company</td>
			<td>Catalog Number</td>
			<td>Comments/Description</td>
		</tr>
		<tr>
			<td>Acetonitrile</td>
			<td>Sigma</td>
			<td>34851</td>
			<td></td>
		</tr>
		<tr>
			<td>Amicon Ultra 4 ml centrifugal filter (3k MWCO)</td>
			<td>Fisher Scientific</td>
			<td>UFC800308 (8pk)</td>
			<td></td>
		</tr>
		<tr>
			<td>Aminopeptidase</td>
			<td>Creative Biomart</td>
			<td>Aminopeptidase-86A</td>
			<td></td>
		</tr>
		<tr>
			<td>Ammonium bicarbonate</td>
			<td>Sigma</td>
			<td>A6141</td>
			<td></td>
		</tr>
		<tr>
			<td>Ammonium sulfate</td>
			<td>Sigma</td>
			<td>A5132</td>
			<td></td>
		</tr>
		<tr>
			<td>Borane-dimethylamine complex</td>
			<td>Sigma</td>
			<td>180238</td>
			<td>Make fresh 1 M solution before use.</td>
		</tr>
		<tr>
			<td>Boric acid</td>
			<td>Sigma</td>
			<td>B6768</td>
			<td></td>
		</tr>
		<tr>
			<td>Bovine pancreas insulin</td>
			<td>Sigma</td>
			<td>I5500</td>
			<td></td>
		</tr>
		<tr>
			<td>C18 Spin Columns</td>
			<td>Thermo Scientific</td>
			<td>89870</td>
			<td></td>
		</tr>
		<tr>
			<td>Deuterium oxide</td>
			<td>Cambridge Isotope Laboratories</td>
			<td>DLM-4-100</td>
			<td></td>
		</tr>
		<tr>
			<td>1,2-dichloroethane-13C2</td>
			<td>Sigma</td>
			<td>714321</td>
			<td>80 mM stock solution stored at -80 &#176;C. Used as reference in NMR spectra at 24 mM.</td>
		</tr>
		<tr>
			<td>2,5-dihydroxybenzoic acid</td>
			<td>Acr&#333;s Organics</td>
			<td>165200050</td>
			<td></td>
		</tr>
		<tr>
			<td>Formaldehyde (36.5% wt/wt)</td>
			<td>Sigma</td>
			<td>33220</td>
			<td>Make fresh 1 M solution before use.</td>
		</tr>
		<tr>
			<td>13C-formaldehyde (99%)</td>
			<td>Cambridge Isotope Laboratories</td>
			<td>CLM-806-1</td>
			<td>Make fresh 1 M solution before use.</td>
		</tr>
		<tr>
			<td>Lysozyme (hen egg white)</td>
			<td>Sigma</td>
			<td>L6376</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium phosphate (dibasic heptahydrate)</td>
			<td>Sigma</td>
			<td>S9390</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium phosphate (monobasic)</td>
			<td>Sigma</td>
			<td>S9638</td>
			<td></td>
		</tr>
		<tr>
			<td>Tricine</td>
			<td>Sigma</td>
			<td>T0377</td>
			<td></td>
		</tr>
		<tr>
			<td>Trifluoroacetic acid</td>
			<td>Sigma</td>
			<td>302031</td>
			<td></td>
		</tr>
		<tr>
			<td>700 MHz Varian VNMRS with 5 mm HCN 5922 probe</td>
			<td>Agilent Technologies</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Bruker UltrafleXtreme MALDI TOF/TOF</td>
			<td>Bruker</td>
			<td></td>
			<td></td>
		</tr>
	</tbody>
</table>

methods to identify the nmr resonances of the 13c-dimethyl n-terminal amine on reductively methylated proteins

Nuclear magnetic resonance (NMR) spectroscopy is a proven technique for protein structure and dynamic studies. To study proteins with NMR, stable magnetic isotopes are typically incorporated metabolically to improve the sensitivity and allow for sequential resonance assignment. Reductive 13C-methylation is an alternative labeling method for proteins that are not amenable to bacterial host over-expression, the most common method of isotope incorporation. Reductive 13C-methylation is a chemical reaction performed under mild conditions that modifies a protein&#39;s primary amino groups (lysine &#949;-amino groups and the N-terminal &#945;-amino group) to 13C-dimethylamino groups. The structure and function of most proteins are not altered by the modification, making it a viable alternative to metabolic labeling. Because reductive 13C-methylation adds sparse, isotopic labels, traditional methods of assigning the NMR signals are not applicable. An alternative assignment method using mass spectrometry (MS) to aid in the assignment of protein 13C-dimethylamine NMR signals has been developed. The method relies on partial and different amounts of 13C-labeling at each primary amino group. One limitation of the method arises when the protein&#39;s N-terminal residue is a lysine because the &#945;- and &#949;-dimethylamino groups of Lys1 cannot be individually measured with MS. To circumvent this limitation, two methods are described to identify the NMR resonance of the 13C-dimethylamines associated with both the N-terminal &#945;-amine and the side chain &#949;-amine. The NMR signals of the N-terminal &#945;-dimethylamine and the side chain &#949;-dimethylamine of hen egg white lysozyme, Lys1, are identified in 1H-13C heteronuclear single-quantum coherence spectra.

Reductive Methylation and pH
A pH induced chemoselective reductive 13C-methylation reaction has been demonstrated on lysozyme. At low pH, the reaction prefers the N-terminal &#945;-amine over the side chain &#949;-amines and vice versa at high pH. In solution the protein&#39;s amino groups exist in equilibrium between the free amine and the conjugate acid, which is tunable with pH. The optimum pH range for this reaction depends on the reducing agent used. In this case, dimethy...

Watch this Scientific Journal Video about Methods to Identify the NMR Resonances of the 13C-Dimethyl N-terminal Amine on Reductively Methylated Proteins at JoVE.com

Methods to Identify the NMR Resonances of the 13C-Dimethyl N-terminal Amine on Reductively Methylated Proteins

Two methods for assigning the &#945;- and &#949;-dimethylamine nuclear magnetic resonance signals of a reductively 13C-methylated N-terminal lysine are described. One method utilizes the pH-induced selectivity of the reductive methylation reaction, and the other uses aminopeptidase to selectively remove the N-terminal lysine.

Methods to Identify the NMR Resonances of the 13C-Dimethyl N-terminal Amine on Reductively Methylated Proteins

Nuclear magnetic resonance (NMR) spectroscopy is a proven technique for protein structure and dynamic studies. To study proteins with NMR, stable magnetic ...

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Chemistry

6.2K visualizações.  Louisiana State University. Two methods for assigning the α- and ε-dimethylamine nuclear magnetic resonance signals of a reductively 13C-methylated N-terminal lysine are described. One method utilizes the pH-induced selectivity of the reductive methylation reaction, and the other uses aminopeptidase to selectively remove the N-terminal lysine.

Vídeo: Methods to Identify the NMR Resonances of the 13C-Dimethyl N-terminal Amine on Reductively Methylated Proteins

Structure and Coordination Determination of Peptide-metal Complexes Using 1D and 2D 1H NMR

Copper (I) binding by metallochaperone transport proteins prevents copper oxidation and release of the toxic ions that may participate in harmful redox reactions. The Cu (I) complex of the peptide model of a Cu (I) binding metallochaperone protein, which includes the sequence MTCSGCSRPG (underlined is conserved), was determined in solution under inert conditions by NMR spectroscopy.
NMR is a widely accepted technique for the determination of solution structures of proteins and peptides. Due to difficulty in crystallization to provide single crystals suitable for X-ray crystallography, the NMR technique is extremely valuable, especially as it provides information on the solution state rather than the solid state. Herein we describe all steps that are required for full three-dimensional structure determinations by NMR. The protocol includes sample preparation in an NMR tube, 1D and 2D data collection and processing, peak assignment and integration, molecular mechanics calculations, and structure analysis. Importantly, the analysis was first conducted without any preset metal-ligand bonds, to assure a reliable structure determination in an unbiased manner.

Structure and Coordination Determination of Peptide-metal Complexes Using 1D and 2D 1H NMR

The NMR-solution structure of a metallochaperone model peptide with Cu (I) was determined, and a detailed protocol from sample preparation and 1D and 2D data collection to a three-dimensional structure is described.

Copper (I) binding by metallochaperone transport proteins prevents copper oxidation and release of the toxic ions that may participate in harmful redox ...

Determining the Ice-binding Planes of Antifreeze Proteins by Fluorescence-based Ice Plane Affinity

Antifreeze proteins (AFPs) are expressed in a variety of cold-hardy organisms to prevent or slow internal ice growth. AFPs bind to specific planes of ice through their ice-binding surfaces. Fluorescence-based ice plane affinity (FIPA) analysis is a modified technique used to determine the ice planes to which the AFPs bind. FIPA is based on the original ice-etching method for determining AFP-bound ice-planes. It produces clearer images in a shortened experimental time. In FIPA analysis, AFPs are fluorescently labeled with a chimeric tag or a covalent dye then slowly incorporated into a macroscopic single ice crystal, which has been preformed into a hemisphere and oriented to determine the a- and c-axes. The AFP-bound ice hemisphere is imaged under UV light to visualize AFP-bound planes using filters to block out nonspecific light. Fluorescent labeling of the AFPs allows real-time monitoring of AFP adsorption into ice. The labels have been found not to influence the planes to which AFPs bind. FIPA analysis also introduces the option to bind more than one differently tagged AFP on the same single ice crystal to help differentiate their binding planes. These applications of FIPA are helping to advance our understanding of how AFPs bind to ice to halt its growth and why many AFP-producing organisms express multiple AFP isoforms.

Antifreeze proteins (AFPs) bind to specific planes of ice to prevent or slow ice growth. Fluorescence-based ice plane affinity (FIPA) analysis is a modification of the original ice-etching method for determination of AFP-bound ice planes. AFPs are fluorescently labeled, incorporated into macroscopic single ice crystals, and visualized under UV light.

Antifreeze proteins (AFPs) are expressed in a variety of cold-hardy organisms to prevent or slow internal ice growth. AFPs bind to specific planes of ice ...

Optimization of Synthetic Proteins: Identification of Interpositional Dependencies Indicating Structurally and/or Functionally Linked Residues

Protein alignments are commonly used to evaluate the similarity of protein residues, and the derived consensus sequence used for identifying functional units (e.g., domains). Traditional consensus-building models fail to account for interpositional dependencies &#8211; functionally required covariation of residues that tend to appear simultaneously throughout evolution and across the phylogentic tree. These relationships can reveal important clues about the processes of protein folding, thermostability, and the formation of functional sites, which in turn can be used to inform the engineering of synthetic proteins. Unfortunately, these relationships essentially form sub-motifs which cannot be predicted by simple &#8220;majority rule&#8221; or even HMM-based consensus models, and the result can be a biologically invalid &#8220;consensus&#8221; which is not only never seen in nature but is less viable than any extant protein. We have developed a visual analytics tool, StickWRLD, which creates an interactive 3D representation of a protein alignment and clearly displays covarying residues. The user has the ability to pan and zoom, as well as dynamically change the statistical threshold underlying the identification of covariants. StickWRLD has previously been successfully used to identify functionally-required covarying residues in proteins such as Adenylate Kinase and in DNA sequences such as endonuclease target sites.

Synthetic protein sequences based on consensus motifs typically ignore co-evolving residues, that imply interpositional dependencies (IPDs). IPDs can be essential to activity, and designs that disregard them may result in suboptimal results. This protocol uses StickWRLD to identify IPDs and help inform rational protein design, resulting in more efficient results.

Protein alignments are commonly used to evaluate the similarity of protein residues, and the derived consensus sequence used for identifying functional ...

Photochemical Oxidative Growth of Iridium Oxide Nanoparticles on CdSe@CdS Nanorods

We demonstrate a procedure for the photochemical oxidative growth of iridium oxide catalysts on the surface of seeded cadmium selenide-cadmium sulfide (CdSe@CdS) nanorod photocatalysts. Seeded rods are grown using a colloidal hot-injection method and then moved to an aqueous medium by ligand exchange. CdSe@CdS nanorods, an iridium precursor and other salts are mixed and illuminated. The deposition process is initiated by absorption of photons by the semiconductor particle, which results with formation of charge carriers that are used to promote redox reactions. To insure photochemical oxidative growth we used an electron scavenger. The photogenerated holes oxidize the iridium precursor, apparently in a mediated oxidative pathway. This results in the growth of high quality crystalline iridium oxide particles, ranging from 0.5 nm to about 3 nm, along the surface of the rod. Iridium oxide grown on CdSe@CdS heterostructures was studied by a variety of characterization methods, in order to evaluate its characteristics and quality. We explored means for control over particle size, crystallinity, deposition location on the CdS rod, and composition. Illumination time and excitation wavelength were found to be key parameters for such control. The influence of different growth conditions and the characterization of these heterostructures are described alongside a detailed description of their synthesis. Of significance is the fact that the addition of iridium oxide afforded the rods astounding photochemical stability under prolonged illumination in pure water (alleviating the requirement for hole scavengers).

A protocol for the photochemical oxidative growth of small crystalline iridium oxide nanoparticles on the surface of CdSe@CdS seeded rod nanoparticles is presented.

We demonstrate a procedure for the photochemical oxidative growth of iridium oxide catalysts on the surface of seeded cadmium selenide-cadmium sulfide ...

Epitaxial Growth of Perovskite Strontium Titanate on Germanium via Atomic Layer Deposition

Atomic layer deposition (ALD) is a commercially utilized deposition method for electronic materials. ALD growth of thin films offers thickness control and conformality by taking advantage of self-limiting reactions between vapor-phase precursors and the growing film. Perovskite oxides present potential for next-generation electronic materials, but to-date have mostly been deposited by physical methods. This work outlines a method for depositing SrTiO3 (STO) on germanium using ALD. Germanium has higher carrier mobilities than silicon and therefore offers an alternative semiconductor material with faster device operation. This method takes advantage of the instability of germanium's native oxide by using thermal deoxidation to clean and reconstruct the Ge (001) surface to the 2&#215;1 structure. 2-nm thick, amorphous STO is then deposited by ALD. The STO film is annealed under ultra-high vacuum and crystallizes on the reconstructed Ge surface. Reflection high-energy electron diffraction (RHEED) is used during this annealing step to monitor the STO crystallization. The thin, crystalline layer of STO acts as a template for subsequent growth of STO that is crystalline as-grown, as confirmed by RHEED. In situ X-ray photoelectron spectroscopy is used to verify film stoichiometry before and after the annealing step, as well as after subsequent STO growth. This procedure provides framework for additional perovskite oxides to be deposited on semiconductors via chemical methods in addition to the integration of more sophisticated heterostructures already achievable by physical methods.

This work details the procedures for the growth and characterization of crystalline SrTiO3 directly on germanium substrates by atomic layer deposition. The procedure illustrates the ability of an all-chemical growth method to integrate oxides monolithically onto semiconductors for metal-oxide semiconductor devices.

Atomic layer deposition (ALD) is a commercially utilized deposition method for electronic materials. ALD growth of thin films offers thickness control and ...

Spin Saturation Transfer Difference NMR (SSTD NMR): A New Tool to Obtain Kinetic Parameters of Chemical Exchange Processes

This detailed protocol describes the new Spin Saturation Transfer Difference Nuclear Magnetic Resonance protocol (SSTD NMR), recently developed in our group to study processes of mutual-site chemical exchange that are difficult to analyze by traditional methods. As the name suggests, this method combines the Spin Saturation Transfer method used for small molecules, with the Saturation Transfer Difference (STD) NMR method employed for the study of protein-ligand interactions, by measuring transient spin saturation transfer along increasing saturation times (build-up curves) in small organic and organometallic molecules undergoing chemical exchange.
Advantages of this method over existing ones are: there is no need to reach coalescence of the exchanging signals; the method can be applied as long as one signal of the exchanging sites is isolated; there is no need to measure T1 or reach steady state saturation; rate constant values are measured directly, and T1 values are obtained in the same experiment, using only one set of experiments.
To test the method, we have studied the dynamics of the hindered rotation of N,N-dimethylamides, for which much data is available for comparison. The thermodynamic parameters obtained using SSTD are very similar to the reported ones (spin-saturation transfer techniques and line-shape analysis). The method can be applied to more challenging substrates that cannot be studied by previous methods.
We envisage that the simple experimental set up and the wide applicability of the method to a great variety of substrates will make this a common technique amongst organic and organometallic chemists without extensive expertise in NMR.

Spin Saturation Transfer Difference NMR SSTD NMR: A New Tool to Obtain Kinetic Parameters of Chemical Exchange Processes

A detailed protocol describing the SSTD NMR method is presented here to help new users apply this new method to obtain the kinetic parameters of their own systems undergoing chemical exchange.

This detailed protocol describes the new Spin Saturation Transfer Difference Nuclear Magnetic Resonance protocol (SSTD NMR), recently developed in our ...

NMR Spectroscopy as a Robust Tool for the Rapid Evaluation of the Lipid Profile of Fish Oil Supplements

The western diet is poor in n-3 fatty acids, therefore the consumption of fish oil supplements is recommended to increase the intake of these essential nutrients. The objective of this work is to demonstrate the qualitative and quantitative analysis of encapsulated fish oil supplements using high-resolution 1H and 13C NMR spectroscopy utilizing two different NMR instruments; a 500 MHz and an 850 MHz instrument. Both proton (1H) and carbon (13C) NMR spectra can be used for the quantitative determination of the major constituents of fish oil supplements. Quantification of the lipids in fish oil supplements is achieved through integration of the appropriate NMR signals in the relevant 1D spectra. Results obtained by 1H and 13C NMR are in good agreement with each other, despite the difference in resolution and sensitivity between the two nuclei and the two instruments. 1H NMR offers a more rapid analysis compared to 13C NMR, as the spectrum can be recorded in less than 1 min, in contrast to 13C NMR analysis, which lasts from 10 min to one hour. The 13C NMR spectrum, however, is much more informative. It can provide quantitative data for a greater number of individual fatty acids and can be used for determining the positional distribution of fatty acids on the glycerol backbone. Both nuclei can provide quantitative information in just one experiment without the need of purification or separation steps. The strength of the magnetic field mostly affects the 1H NMR spectra due to its lower resolution with respect to 13C NMR, however, even lower cost NMR instruments can be efficiently applied as a standard method by the food industry and quality control laboratories.

Here, high-resolution 1H and 13C Nuclear Magnetic Resonance (NMR) spectroscopy was used as a rapid and reliable tool for quantitative and qualitative analysis of encapsulated fish oil supplements.

The western diet is poor in n-3 fatty acids, therefore the consumption of fish oil supplements is recommended to increase the intake of these essential ...

Application and Methodology of the Non-destructive 19F Time-domain NMR Technique to Measure the Content in Fluorine-containing Drug Products

Here, we describe a protocol developed by our group that uses low-field fluorine-19 (19F) time-domain (TD) nuclear magnetic resonance (NMR) to measure the average content of fluorinated drugs in their formulated drug product forms: tablets or capsules. This method is specific to fluorinated drugs because it detects only the content of fluorine, avoiding interference from the excipients that lack fluorine. The advantages of measuring the active content of fluorinated drugs using low-field 19F TD-NMR versus high-field 19F solid-state (SS) NMR are the simplicity of the method; the low cost; and the non-destructive nature of the technique, with all samples recoverable in intact forms (e.g., powders, tablets, and capsules), making this technique affordable for any laboratory.
We have tested the method with three fluorinated drug products available on the market - cinacalcet, lansoprazole, and ciprofloxacin - with doses ranging from 15 to 500 mg. The results of the analyses, measured by low-field 19F TD-NMR, supported the reported label claims for the average drug content.
Based on the simplicity and reproducibility of the analysis, we envision this methodology being implemented in any laboratory, including manufacturing plants, as a process analytical technology (PAT) tool in the pharmaceutical industry.

Application and Methodology of the Non-destructive 19F Time-domain NMR Technique to Measure the Content in Fluorine-containing Drug Products

A simple and non-destructive technique that measures the average content of drug substances in formulated drug products containing fluorine using low-field fluorine-19 (19F) time-domain (TD) nuclear magnetic resonance (NMR) is presented here. The technique can be applied to the development and manufacturing of drugs in the pharmaceutical industry.

Here, we describe a protocol developed by our group that uses low-field fluorine-19 (19F) time-domain (TD) nuclear magnetic resonance (NMR) to measure the ...

Raman and IR Spectroelectrochemical Methods as Tools to Analyze Conjugated Organic Compounds

In the presented work, two spectroelectrochemical techniques are discussed as tools for the analysis of the structural changes occurring in the molecule on the vibrational level of energy. Raman and IR spectroelectrochemistry can be used for advanced characterization of the structural changes in the organic electroactive compounds. Here, the step-by-step analysis by means of Raman and IR spectroelectrochemistry is shown. Raman and IR spectroelectrochemical techniques provide complementary information about structural changes occurring during an electrochemical process, i.e. allows for the investigation of redox processes and their products. The examples of IR and Raman spectroelectrochemical analysis are presented, in which the products of the redox reactions, both in solution and solid state, are identified.

A protocol of step-by-step Raman and IR spectroelectrochemical analysis is presented.

In the presented work, two spectroelectrochemical techniques are discussed as tools for the analysis of the structural changes occurring in the molecule ...

Electrochemical Preparation of Poly(3,4-Ethylenedioxythiophene) Layers on Gold Microelectrodes for Uric Acid-Sensing Applications

Two different methods for the synthesis of poly(3,4-ethylenedioxythiophene) (PEDOT) on gold electrodes are described, using electropolymerization of 3,4-ethylenedioxythiophene (EDOT) monomer in an aqueous and an organic solution. Cyclic voltammetry (CV) was used in the synthesis of PEDOT thin layers. Lithium perchlorate (LiClO4) was used as a dopant in both aqueous (aqueous/acetonitrile (ACN)) and organic (propylene carbonate (PC)) solvent systems. After the PEDOT layer was created in the organic system, the electrode surface was acclimatized by successive cycling in an aqueous solution for use as a sensor for aqueous samples. 
The use of an aqueous-based electropolymerization method has the potential benefit of removing the acclimatization step to have a shorter sensor preparation time. Although the aqueous method is more economical and environmentally friendly than the organic solvent method, superior PEDOT formation is obtained in the organic solution. The resulting PEDOT electrode surfaces were characterized by scanning electron microscopy (SEM), which showed the constant growth of PEDOT during electropolymerization from the organic PC solution, with rapid fractal-type growth on gold (Au) microelectrodes.

Electrochemical Preparation of Poly3,4-Ethylenedioxythiophene Layers on Gold Microelectrodes for Uric Acid-Sensing Applications

We describe aqueous and organic solvent systems for the electropolymerization of poly(3,4-ethylenedioxythiophene) to create thin layers on the surface of gold microelectrodes, which are used for sensing low molecular weight analytes.

Two different methods for the synthesis of poly(3,4-ethylenedioxythiophene) (PEDOT) on gold electrodes are described, using electropolymerization of ...