Effect of methylation on the side-chain pKa value of arginine

Abstract

Arginine methylation is important in biological systems. Recent studies link the deregulation of protein arginine methyltransferases with certain cancers. To assess the impact of methylation on interaction with other biomolecules, the pKa values of methylated arginine variants were determined using NMR data. The pKa values of monomethylated, symmetrically dimethylated, and asymmetrically dimethylated arginine are similar to the unmodified arginine (14.2 ± 0.4). Although the pKa value has not been significantly affected by methylation, consequences of methylation include changes in charge distribution and steric effects, suggesting alternative mechanisms for recognition.

Keywords: NMR spectroscopy; arginine; methylation; methyltransferase; pH titration; pKa determination.

Figure 1

A: Arginine and methylated arginine variants. N‐ and C‐termini contain protection groups to mimic a peptide environment. B: ¹H NMR (600 MHz) of the delta protons of 0.5 mM MMA at 25°C from pH 13.6 to 11.1 (top to bottom). C: ¹H NMR pH titration of aDMA (black), sDMA (red), MMA (green), and protected control arginine (blue) delta protons.

Figure 2

Titration curves for (A) control compounds and (B) the protected control arginine. Monitoring the chemical shift and pH for (A) H₂ of imidazole (top) and trifluoroethanol (bottom) and (B) H_δ of arginine with a simulated curve for a hypothetical pK _a of 12.48 (red trace). Experimental data are represented by circles for imidazole (A) and by diamonds for trifluoroethanol and (B) blue triangles for arginine. The lines are for simulated curves, assuming a difference in chemical shift between the protonated and unprotonated species of 0.2 (dotted), 0.4 (solid), and 0.6 (dashed) ppm for the arginine variants, and 0.4, 0.5, and 0.6 ppm for imidazole.

Figure 3

Titration curves for ¹H_δ and ¹H_methyl as a function of pH for 0.5 mM (A and B) aDMA, (C and D) sDMA, and (E and F) MMA. For each plot, experimental data are represented by data points, closed for delta proton chemical shifts and open for methyl proton chemical shifts, solid black lines are for estimated unprotonated species (0.4 ppm difference from the protonated species), dotted lines are for estimated unprotonated species (0.2 ppm difference from the protonated species), and dashed lines are for unprotonated species (0.6 ppm difference from the protonated species). Data were collected on a 5‐mm QXI probe at 600 MHz.

Figure 4

Titration curves for the control arginine for natural abundance (A) ¹³C_ζ and (B) ¹³C_γ chemical shifts for 50 mM control arginine with additional amide group at the C‐terminus and acetyl at the N‐terminus to mimic a peptide environment. Because degradation at the backbone was observed, data points above pH 14.04 were not recorded. The Δδ used were from previously reported values of 4 and 1 ppm,24 yielding pK _a values of 13.8 and 14.1, respectively. Data were collected using a 10‐mm BBO probe at 150.9 MHz.

Figure 5

Electrostatic potential maps of guanidinium head groups calculated using Spartan 10. The constructs shown above depict representative shortened, model structures of the four arginine variants: (A) unmethylated, (B) MMA, (C) aDMA, and (D) sDMA. The color scale for the electric potentials is shown in kilojoules per mole, where red represents the lowest electrostatic potential (electron rich) and blue represents the highest electrostatic potential (electron poor) regions. Tautomers for each variant were constructed and individually minimized; the lowest energy tautomer is shown.