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 Prepare 500 ml of a 50 mM sodium phosphate buffer at pH 7.5. Store at room temperature. Label three 1 ml microcentrifuge tubes for fully labeled sample (FLS), low pH partially labeled sample (LpH), and high pH partially labeled sample (HpH). 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 deg…

Representative 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, dimethy…

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</sup…

Materials

Name of Material/ Equipment	Company	Catalog Number	Comments/Description
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.
2,5-dihydroxybenzoic acid	Acrōs Organics	165200050
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