Name: methods to identify the nmr resonances of the 13c-dimethyl n-terminal amine on reductively methylated proteins
Uploaded: 2013-12-12T13:45:00
Description: 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

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

1. Reductive Methylation of Lysozyme
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	<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 deg...

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Reson. Shifts..</a> J. Biol. Chem. 258 (14), 8663-8669 (1983).</li><li>Dick, L., Sherry, A., Newkirk, M., Gray, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Reductive+Methylation+and+C-13+NMR+Studies+of+the+Lysyl+Residues+of+fd+Gene+5+Protein+-+Lysines+24,+46,+and+69+May+be+Involved+in+Nucleic+Acid+Binding..">Reductive Methylation and C-13 NMR Studies of the Lysyl Residues of fd Gene 5 Protein - Lysines 24, 46, and 69 May be Involved in Nucleic Acid Binding..</a> J. Biol. Chem. 263 (35), 18864-18872 .</li><li>Moore, G. R., 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=N-epsilon,N-epsilon-dimethyl-lysine+cytochrome+c+as+an+NMR+probe+for+lysine+involvement+in+protein-protein+complex+formation.">N-epsilon,N-epsilon-dimethyl-lysine cytochrome c as an NMR probe for lysine involvement in protein-protein complex formation.</a> Biochem. J. 332, 439-449 (1998).</li><li>Jentoft, J. E., Gerken, T. A., Jentoft, N., 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=Studies+of+the+Active-Site+Lysine+of+Ribonuclease-A+By+C-13+NMR..">Studies of the Active-Site Lysine of Ribonuclease-A By C-13 NMR..</a> Biophys. J. 25 (2), 56 (1979).</li><li>Gerken, T., Jentoft, J., Jentoft, N., Dearborn, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Intramolecular+Interactions+of+Amino+Groups+in+C-13+Reductively+Methylated+Hen+Egg-White+Lysozyme.">Intramolecular Interactions of Amino Groups in C-13 Reductively Methylated Hen Egg-White Lysozyme.</a> J. Biol. Chem. 257 (6), 2894-2900 (1982).</li><li>Dick, L. R., Geraldes, C., Sherry, A. D., Gray, C. W., Gray, D. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=C-13+NMR+of+Methylated+Lysines+of+fd+Gene-5+Protein+-+Evidence+for+A+Conformational+Change+Involving+Lysine-24+Upon+Binding+of+A+Negatively+Charged+Lanthanide+Chelate.">C-13 NMR of Methylated Lysines of fd Gene-5 Protein - Evidence for A Conformational Change Involving Lysine-24 Upon Binding of A Negatively Charged Lanthanide Chelate.</a> Biochem. 28 (19), 7896-7904 (1989).</li><li>Gluck, M., Sweeney, W. 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Chem. 267 (36), 25830-25838 (1992).</li><li>Ashfield, J., 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=Chemical+Modification+of+a+Variant+of+Human+MIP-1+Alpha,+Implications+for+Dimer+Structure..">Chemical Modification of a Variant of Human MIP-1 Alpha, Implications for Dimer Structure..</a> Protein Science. 9 (10), 2047-2053 (2000).</li><li>Goux, W., Teherani, J., Sherry, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Amine+Inversion+in+Proteins+-+A+C-13-NMR+Study+of+Proton+Exchange+and+Nitrogen+Inversion+Rates+in+N-Epsilon,N-Epsilon,N-Alpha,N-Alpha-[C-13]Tetramethyllysine,+N-Epsilon,N-Epsilon,N-Alpha,N-Alpha[C-13]Tetramethyllysine+Methyl-Ester,+and+Reductively+Methylated+Concanavalin-A..">Amine Inversion in Proteins - A C-13-NMR Study of Proton Exchange and Nitrogen Inversion Rates in N-Epsilon,N-Epsilon,N-Alpha,N-Alpha-[C-13]Tetramethyllysine, N-Epsilon,N-Epsilon,N-Alpha,N-Alpha[C-13]Tetramethyllysine Methyl-Ester, and Reductively Methylated Concanavalin-A..</a> Biophysical Chemistry. 19 (4), 363-373 (1984).</li><li>Sherry, A. D., Keepers, J., James, T. L., Teherani, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Methyl+Motions+in+C-13+Methylated+Concanavalin-as+Studied+by+C-13+Magnetic-Resonance+Relaxation+Techniques.">Methyl Motions in C-13 Methylated Concanavalin-as Studied by C-13 Magnetic-Resonance Relaxation Techniques.</a> Biochemistry. 23 (14), 3181-3185 (1984).</li><li>Huque, M., Vogel, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=C-13+NMR+Studies+of+the+Lysine+Side+Chains+of+Calmodulin+and+Its+Proteolytic+Fragments.">C-13 NMR Studies of the Lysine Side Chains of Calmodulin and Its Proteolytic Fragments.</a> Journal of Protein Chemistry. 12 (6), 695-707 (1993).</li><li>Brown, L., Bradbury, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Proton-Magnetic-Resonance+Studies+of+Lysine+Residues+of+Ribonuclease-A.">Proton-Magnetic-Resonance Studies of Lysine Residues of Ribonuclease-A.</a> Eur. 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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.

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	<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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