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

핵 자기 공명 (NMR) 분광법은 단백질 구조 및 동적 연구 입증 된 기술이다. NMR로 단백질을 연구하기 위해, 안정적인 자기 동위 원소는 일반적으로 감도를 개선하고 순차적 공명 할당을 허용 대사 포함된다. 환원 13 C-메틸화를 통해 표현 박테리아 숙주, 동위 원소 결합하는 가장 일반적인 방법에 순종하지 않는 단백질에 대한 대체 표시 방법입니다. 환원성 13 C-메틸화는 13 C-디메틸 아미노 그룹으로의 단백질의 일차 아미노기 (라이신의 ε-아미노기와 N-말단 α-아미노기를) 수정 온화한 조건 하에서 수행 화학 반응이다. 대부분의 단백질의 구조와 기능은 그 대사 라벨링에 대한 실행 가능한 대안 만들기, 수정에 의해 변경되지 않습니다. 환원 13 C-메틸화는 스파 스, 동위 원소 라벨, N을 할당하는 전통적인 방법을 추가하기 때문에MR 신호는 적용되지 않습니다. 단백질 (13) C-디메틸 NMR 신호의 할당을 돕기 위해 질량 분석기 (MS)를 사용하여 대안의 할당 방법이 개발되었다. 이 방법은 각각의 차 아미노기에 13 C-라벨의 부분과 다른 양에 의존한다. Lys1의 α-및 ε-다이 메틸 아미노 그룹이 개별적으로 MS로 측정 할 수 없기 때문에, 단백질의 N-말단 잔기가 리신 때 방법의 제한이 발생한다. 이 한계를 회피하기 위해,이 방법은 N-말단 α-아민 및 측쇄 ε-아민 모두와 연관된 13 C-NMR dimethylamines의 공진을 식별하는 기술된다. N-말단 α-디메틸 아민과 암탉 흰자 리소자임, Lys1의 사이드 체인 ε-디메틸 아민의 NMR 신호는 1 H-13 C 헤테로 핵 단일 양자 간섭 스펙트럼에서 확인된다.

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.

의 NMR 공명 확인하는 방법<sup&gt; 13</sup환원 메틸을 단백질에&gt; C-디메틸 N-말단 아민

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

Research

JoVE Journal

Chemistry

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

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 ...

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

형광 기반 얼음 비행기 선호도에 의해 부동액 단백질의 얼음 결합 평면 결정

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

합성 단백질의 최적화 : Interpositional 종속 관계 확인이 구조적으로 표시 및 / 또는 기능적으로 연결된 잔류

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

Preparation of Highly Porous Coordination Polymer Coatings on Macroporous Polymer Monoliths for Enhanced Enrichment of Phosphopeptides

포스 포의 향상된 농축을위한 거대 다공성 고분자 모노리스에 다공성 조정 폴리머 코팅의 제조

We describe a protocol for the preparation of hybrid materials based on highly porous coordination polymer coatings on the internal surface of macroporous ...

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

의 CdSe @ CdS와 나노 막대에 이리듐 산화물 나노 입자의 광 화학적 산화 성장

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

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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

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

스핀 채도 전송 차이 NMR (SSTD NMR) : 새로운 도구 화학 Exchange 프로세스의 키네틱 매개 변수를 구하는

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

생선 기름 보충제의 지질 프로필의 신속한 평가를위한 강력한 도구로 NMR 분광학

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

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

응용 프로그램 및 비 파괴적인 19F 시간 영역 NMR 기법의 방법론 포함 하는 불 소 약 제품에 콘텐츠를 측정 하

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

라만 방법과 IR Spectroelectrochemical 도구로 활용 된 유기 화합물을 분석 하

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