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

Summary

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.

Abstract

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's primary amino groups (lysine ε-amino groups and the N-terminal α-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's N-terminal residue is a lysine because the α- and ε-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 α-amine and the side chain ε-amine. The NMR signals of the N-terminal α-dimethylamine and the side chain ε-dimethylamine of hen egg white lysozyme, Lys1, are identified in ¹H-¹³C heteronuclear single-quantum coherence spectra.

Introduction

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 host^2-3. However, two disadvantages of bacterial host over-expression are it cannot produce post-translational modifications and does not work for all proteins^3-4. 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 acids^7-9. 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 α-amine and the lysine, side chain ε-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 temperatures^11,12. In the presence of formaldehyde and a reducing agent, such as dimethylamine borane complex (DMAB), the lysine ε-amino groups and the N-terminal α-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 agent^13,14.

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 Ź⁶. 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 β₂ adrenergic receptor¹⁷, ribonuclease A¹⁸, lysozyme^19, 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, and MIP-1α²⁸ The dynamics of the ¹³C-dimethylamino groups have been studied on concanavalin A^29-30 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 properties^{11,19,23,26,31,32}. 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-1α 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-1α 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'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 α- and ε-¹³C-dimethylamines cannot be independently measured.

Here we present two methods, one chemical and one enzymatic, to identify the N-terminal α-dimethylamine and the side chain ε-dimethylamine sites of an N-terminal lysine residue. The first method was inspired by Córdova et al. who used pH to control the selectivity of protein acetylation³⁵. The reaction favors the N-terminal α-amine at low pH and the side chain ε-amines at high pH, allowing the protein α- and ε-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 α- and ε-¹³C-dimethylamines takes advantage of the selective removal of the N-terminal residue(s) using recombinant Aeromonas proteolytica aminopeptidase.

Protocol

1. Reductive Methylation of Lysozyme

1. Prepare 500 ml of a 50 mM sodium phosphate buffer at pH 7.5. Store at room temperature.
2. Label three 1 ml microcentrifuge tubes for fully labeled sample (FLS), low pH partially labeled sample (LpH), and high pH partially labeled sample (HpH).
3. 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. Cover the tubes with aluminum foil to protect the formaldehyde from degradation due light exposure. Add 500 μl of lysozyme solution to each tube.
4. Prepare fresh 1.0 M solutions of dimethylamine borane complex (DMAB) and ¹³C-formaldehyde. Store at 0 °C.
5. 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 α-¹³C-dimethylamine peak at low pH, a 2:7 ratio was required.
6. Add 6.1 μl of 1.0 M DMAB to FLS, add 3.2 μl of 1.0 M DMAB to LpH and HpH, and gently vortex. Then add 12.3 μl of 1.0 M ¹³C-formaldehyde to FLS and 6.1 μl of 1.0 M ¹³C-formaldehyde to LpH and HpH. Shake the reaction mixtures at 4 °C for 2 hr.
7. Add another aliquot of 1.0 M DMAB and ¹³C-formaldehyde as in the previous step and shake the reaction mixture overnight at 4 °C for a total reaction time of 18-24 hr.
8. Exchange the sample'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).
9. 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.
10. Cover the sample tubes with foil, add 6.1 μl of DMAB to LpH and HpH, and gently vortex. Then add 12.3 μl of natural abundance formaldehyde. Shake the reaction mixtures at 4 °C for 2 hr.
11. 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 °C for a total reaction time of 18-24 hr. Then buffer exchange using steps 2.5-2.6 and store 50 μg samples at -20 °C for MS.

2. Prepare the Sample for NMR Analysis

1. Prepare a 2.0 M sodium borate buffer solution at pH 8.5 in H₂O.
2. Add 375 μl 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.
3. Place the frozen sample on a lyophilizer to dry. Remove when dry.
4. Using a pipette, add 15 ml of D₂O to each tube and mix.
5. 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 μl). 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 μl using a centrifuge with a 35° fixed angle rotor at 4 °C and 7,500 rcf.
6. Empty the trap under each filter. Add another 3.0 ml of buffer to the samples and concentrate the samples again to 500 μl. Repeat step 2.5 4x.
7. 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 μM samples.
8. Transfer 530 μl to a 5 mm NMR tube or 250 μM to a 5 mm magnetic susceptibility matched tube.
9. Prepare an 80 mM stock solution of 1,2-dichloroethane-¹³C₂ in D₂O. Store this stock solution at -80 °C. Dilute to 24 mM in 80 μl 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.

3. Aminopeptidase Degradation of Lysozyme

1. Prepare a 5 mg/ml solution of lysozyme in ultrapure water.
2. Add 250 μl of the lysozyme solution to a clean, 1 ml centrifuge tube.
3. Add 25 μl of a 0.1 M tricine solution, pH 8.0, and 6 μl of a 2.16 μg/ml Aeromonas proteolytica aminopeptidase solution.
4. Incubate at 37 °C for 6 hr.
5. Add an additional 6 μl of the 2.16 μg/ml aminopeptidase solution to the sample for a 1:50 molar ratio of aminopeptidase to lysozyme. Incubate for an addition 20 hr at 37 °C.
6. Desalt 30 μl of the sample using a C18 spin column, (follow manufacturer's protocol). Store at -20 °C for MALDI MS analysis.
7. Prepare the remaining protein sample for NMR as described in protocol 2).

4. Prepare Samples for Mass Spectrometry

1. Using the 50 μg of sample reserved in step 2.3, exchange the buffer to 100 mM ammonium bicarbonate, pH 8.0, and concentrate to approximately 50 μl 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.
2. Add 2.0 μl of 500 mM dithiothreitol to reduce the disulfide bonds. Incubate the solution at 60 °C for 1 hr.
3. Add 2 μl of freshly prepared 500 mM iodoacetamide to alkylate the thiol groups. Incubate at room temperature for 20 min.
4. Remove the foil and add 1.0 μl of the 500 mM dithiothreitol solution to quench the reaction.
5. Add 2.0 μg of sequencing grade trypsin to digest the protein. Incubate the mixture at 37 °C for 16-24 hr.
6. Add TFA until the pH is less than 2 to quench the digestion. (Formic and acetic acids can be used as well)
7. 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.
8. Reconstitute the sample in 5.0 μl 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.

Results

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 α-amine over the side chain ε-amines and vice versa at high pH. In solution the protein'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, dimethylamine borane ...

Discussion

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

Materials

List of materials used in this article

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