Lysine methylation strategies for characterizing protein conformations by NMR

Abstract

In the presence of formaldehyde and a mild reducing agent, reductive methylation is known to achieve near complete dimethylation of protein amino groups under non-denaturing conditions, in aqueous media. Amino methylation of proteins is employed in mass spectrometric, crystallographic, and NMR studies. Where biosynthetic labeling is prohibitive, amino (13)C-methylation provides an attractive option for monitoring folding, kinetics, protein-protein and protein-DNA interactions by NMR. Here, we demonstrate two improvements over traditional (13)C-reductive methylation schemes: (1) By judicious choice of stoichiometry and pH, ε-aminos can be preferentially monomethylated. Monomethyl tags are less perturbing and generally exhibit improved resolution over dimethyllysines, and (2) By use of deuterated reducing agents and (13)C-formaldehyde, amino groups can be labeled with (13)CH(2)D tags. Use of deutero-(13)C-formaldehyde affords either (13)CHD(2), or (13)CD(3) probes depending on choice of reducing agent. Making use of (13)C-(2)H scalar couplings, we demonstrate a filtering scheme that eliminates natural abundance (13)C signal.

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

“…The previous assignments of Lys1 α-and ε-dimethylamine were ambiguous because only the average 13 C %-incorporation was measured 34 . With the pH and aminopeptidase methods, the α-and εdimethylamine peaks can be assigned directly and agree with assignments made by others 23,37,38 . Controlling the pH of the reductive 13 Cmethylation and using aminopeptidase provide two, complementary methods to distinguish the NMR signals of the N-terminal α-and εdimethylamine groups.…”

13C NMR detects conformational change in the 100-kD membrane transporter ClC-ec1

“…The addition of 13 C-enriched formaldehyde to protein under reducing conditions results in the introduction of two 13 C-methyl groups to the ε-amino group of each Lys residue as well as the α-amino group of the N-terminus. The addition of the second methyl group is a more favorable reaction than the addition of the first methyl group, and the reaction favors production of fully dimethylated amines under standard conditions with excess reagents (Larda et al 2012). The NMR spectra of reductively methylated proteins show good signal-to-noise ratio even for large proteins at low protein concentrations (Bokoch et al 2010).…”

“…Because Lys dimethylation preserves charge and introduces only a small steric addition, it has generally been found to be non-perturbing (Larda et al 2012). Nevertheless, the impact of reductive methylation on protein function must be evaluated on a case-by-case basis, with functional comparison of methylated and unmethylated protein.…”

“…However, our strategy to assign resonances by mutagenesis was foiled by the reversion to dimeric stoichiometry upon introduction of additional mutations. As an alternative labeling strategy, we added 13 C methyl groups to Lys residues on WT ClC-ec1, using post-translational reductive methylation (Larda et al 2012; Rayment 1997). The introduction of dimethyl groups onto Lys residues preserves the charge and is generally non-perturbing (Gerken et al 1982; Kurinov et al 2000; Larda et al 2012; Rayment 1997; Rypniewski et al 1993; Walter et al 2006).…”

“…As an alternative labeling strategy, we added 13 C methyl groups to Lys residues on WT ClC-ec1, using post-translational reductive methylation (Larda et al 2012; Rayment 1997). The introduction of dimethyl groups onto Lys residues preserves the charge and is generally non-perturbing (Gerken et al 1982; Kurinov et al 2000; Larda et al 2012; Rayment 1997; Rypniewski et al 1993; Walter et al 2006). The NMR spectra of reductively methylated proteins show good signal-to-noise ratio, with a 13 C methyl signal from the Lys being approximately ten times as intense as the signal from an aliphatic methyl group at similar protein concentrations (Abraham et al 2008).…”