NMR Determination of Protein pK(a) Values in the Solid State

Abstract

Charged residues play an important role in defining key mechanistic features in many biomolecules. Determining the pK(a) values of large, membrane or fibrillar proteins can be challenging with traditional methods. In this study we show how solid-state NMR is used to monitor chemical shift changes during a pH titration for the small soluble β1 immunoglobulin binding domain of protein G. The chemical shifts of all the amino acids with charged side-chains throughout the uniformly-(13)C,(15)N-labeled protein were monitored over several samples varying in pH; pK(a) values were determined from these shifts for E27, D36, and E42, and the bounds for the pK(a) of other acidic side-chain resonances were determined. Additionally, this study shows how the calculated pK(a) values give insights into the crystal packing of the protein.

Figure 1

Overlay of ¹³C-¹³C 2D spectra acquired on uniformly-¹³C,¹⁵N labeled GB1 at pH = 3.63 (blue) and pH = 5.64 (red) using SPC5₃ mixing.

Figure 2

(a) Side-chain carbonyl chemical shifts over the pH titrated range. Expansion of the CG-CO and CD-CO region of the ¹³C-¹³C 2D spectra acquired with SPC5₃ mixing for samples at pH=3.63 (purple), pH=3.95 (blue), pH=4.55 (green), pH=5.22 (orange) and pH=5.64 (red), and pH dependence of ¹³C side-chain carbonyl chemical shifts in nanocrystalline GB1. (b) Aspartic acid CG resonances and (c) glutamic acid CD and C-terminus carbonyl resonances plotted against sample buffer pH showing fitted curves for D36 (blue), E27 (black) and E42 (red).

Figure 3

Three GB1 molecules in trigonal crystal packing (PDB:2QMT) with chemical shift differences between low (pH = 3.63) and high (pH = 5.64) pH samples for the carboxylic acid side-chains of Asn/Asp/Gln/Glu residues colored from no change (blue) to > 2.0 ppm chemical shift differences (red). Residues with > 2.0 ppm chemical shift differences are labeled.