NMR Determination of Protein pK_aValues 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. KeywordsChemical shift perturbation; GB1; Magic-angle spinning; pK a determination; Solid-state NMR Charged residues of a protein play an important role in catalytic reactions, protein stability and substrate binding. [1][2][3] Many enzymatic reactions are controlled by the reversible ionization of charged residues within the active site. This ionization is determined by the intrinsic pK a , which for residues involved in enzymatic mechanisms can deviate greatly from canonical values. 1,[4][5][6] Moreover, pK a values are related to the pH range over which proteins are stable, consistent with the observation of pK a differences among the unfolded and folded proteins, 2,7 with aberrant pK a values often observed in the context of hydrogen bonding, through coupled protonation and deprotonation events. 8 Knowledge of pK a values is useful for purposes of optimizing the absorption and stability of drugs, as well as for identifying substrate interactions in a binding pocket. The central role of ionization events in enzymology has driven the biophysical chemical community to develop methods to accurately determine pK a values across a variety of platforms. 13 Additionally, isotropic chemical shifts were utilized to study rhodopsin and bathorhodopsin and the chromophore retinylidene to gain insights into electrostatic interactions and protonation states in ligation. 14-18 However, these previous SSNMR studies were completed for a small selectively labeled cofactor in the protein systems using one-and two-dimensional experiments, and as such would require many samples in order to assess protonation states of several sites in the protein. Due to the complexity of these issues, no full titration curve to obtain exact pK a values, as routinely done now in solution NMR, has yet been completed utilizing highresolution SSNMR. In this work, we demonstrate that SSNMR can determine both specific pK a values and set bounds for multiple acidic residues in a uniformly labeled protein using multidimensional homo and heteronuclear experiments. The pK a values determined in this work give insight of the protein stability and packing of β1 immunoglobulin binding domain of protein G (GB1) in a nanocrystal environmen...

Site-Specific Internal Motions in GB1 Protein Microcrystals Revealed by 3D 2H–13C–13C Solid-State NMR Spectroscopy

“…However, it is impacted by an intermolecular interaction, where D40 forms a salt bridge with K10 (4.95 Å). 90 , 92 As discussed above for K10, the salt bridge, if it exists, is highly dynamic and only slightly rigidifies the D40 and K10 side chains. The N8 side chain presents moderate flexibility due to the coexistence of opposite effects—being mobilized by the surrounding solvent and potentially restricted by hydrogen bonds ( Figure 3 I).…”

“…This tight packing is also found to be responsible for its COO – p K a value being higher than expected. 92 The values of N37 imply the immobility of the side chain that resides at the edge of the open pocket formed between β strands and the α helix and is partially exposed to solvent ( Figure 3 F). This unique position does not explain the rigidity of the side chain.…”

“…However, an intramolecular salt bridge is likely formed between E15 COO – and K4 (NH3)ζ + as supported by the XRD structure (PDB: 2QMT( 90 )) and E15 pH titration behavior. 92 This seems controversial to the large-amplitude side-chain motions of both E15 and K4. The most likely explanation is that the salt bridge is continually broken and reformed and, therefore, shows negligible impact on the residue side-chain motions.…”

On the use of ultracentrifugal devices for routine sample preparation in biomolecular magic-angle-spinning NMR

“…The time and speed of centrifugation required for pelleting the sample depends on the nature of the sample. Protein crystals and aggregates often have densities that are significantly larger than that of water (1.24 to 1.54 g/mL (Bell et al 1982; White et al 2007)), which facilitates pelleting at relatively moderate g-forces, even in small table top centrifuges (Schmidt et al 2010). Samples featuring lipid LUVs require the use of ultracentrifugation, due to the small density differences (~1.03 g/mL) with the aqueous buffer (see also below).…”

The molecular pH-response mechanism of the plant light-stress sensor PsbS

“…2B), these two peaks fully disappear while gain of signal is observed of shoulders at 179.3 and 177.1 ppm. It is well known that protonation of Glu and Asp induces an upfield shift the carboxyl 13 C NMR resonances 25,26 . We therefore can interpret the signal change as an indication that most of the titratable Asp and Glu residues, 19 in total for P. patens PsbS, are protonated at pH 5.0.…”