pH-dependent random coil 1H, 13C, and 15N chemical shifts of the ionizable amino acids: a guide for protein pK a measurements

Platzer, Gerald; Okon, Mark; McIntosh, Lawrence P.

doi:10.1007/s10858-014-9862-y

Cited by 196 publications

(301 citation statements)

References 214 publications

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“…3C). The pK a value for the retracted state is consistent with the value generally observed for the serine phosphoryl group (21), and is also consistent with the structure of the retracted state in N-HSQC spectra for residues I23, A28, and D32 in pUb and the proximal Ub of pK63-diUb, respectively. The data were collected at 298 K with the proteins prepared in 20 mM Pipes buffer containing 150 mM NaCl.…”

Section: Resultssupporting

confidence: 88%

Ubiquitin S65 phosphorylation engenders a pH-sensitive conformational switch

Gong

et al. 2017

Proc. Natl. Acad. Sci. U.S.A.

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Ubiquitin (Ub) is an important signaling protein. Recent studies have shown that Ub can be enzymatically phosphorylated at S65, and that the resulting pUb exhibits two conformational states-a relaxed state and a retracted state. However, crystallization efforts have yielded only the structure for the relaxed state, which was found similar to that of unmodified Ub. Here we present the solution structures of pUb in both states obtained through refinement against state-specific NMR restraints. We show that the retracted state differs from the relaxed state by the retraction of the last β-strand and by the extension of the second α-helix. Further, we show that at 7.2, the pK a value for the phosphoryl group in the relaxed state is higher by 1.4 units than that in the retracted state. Consequently, pUb exists in equilibrium between protonated and deprotonated forms and between retracted and relaxed states, with protonated/relaxed species enriched at slightly acidic pH and deprotonated/retracted species enriched at slightly basic pH. The heterogeneity of pUb explains the inability of phosphomimetic mutants to fully mimic pUb. The pHsensitive conformational switch is likely preserved for polyubiquitin, as single-molecule FRET data indicate that pH change leads to quaternary rearrangement of a phosphorylated K63-linked diubiquitin. Because cellular pH varies among compartments and changes upon pathophysiological insults, our finding suggests that pH and Ub phosphorylation confer additional target specificities and enable an additional layer of modulation for Ub signals., a 76-residue signaling protein, is found ubiquitously in cells. Two or more Ub molecules can be covalently linked to form a diubiquitin (diUb) and then a polyubiquitin (polyUb), as an isopeptide bond is formed between the carboxylate group of one Ub (called the distal Ub) and the amine group of another Ub (called the proximal Ub). Owing to the characteristic quaternary structures of polyUb and specific interactions between polyUb and its target proteins (1, 2), a polyUb with a specific linkage can be involved in a distinctive set of cellular functions (3).The heterogeneity of Ub also arises from other types of covalent modifications. Proteomics studies have indicated that Ub is phosphorylated at multiple sites (4, 5). However, PINK1 is the only Ub kinase known to date, which specifically phosphorylates Ub at S65 (6, 7). Under normal conditions, only a fraction of Ub is S65-phosphorylated. However, upon oxidative stress, neurodegeneration, or aging, the level of S65 phosphorylation increases significantly (4, 8). S65-phosphorylated Ub (pUb) in turn can activate PARKIN, a ubiquitin ligase, and induce mitophagy (9-12). However, no other pUb-specific targets have been clearly identified, and how phosphorylation affects Ub signaling in general remains unclear.Previously, Komander and coworkers showed that pUb gives two distinct sets of NMR peaks, which correspond to the two conformational states of pUb exchanging at a slow timescale (13). Based on NMR long-r...

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Section: Resultssupporting

confidence: 88%

Ubiquitin S65 phosphorylation engenders a pH-sensitive conformational switch

Gong

et al. 2017

Proc. Natl. Acad. Sci. U.S.A.

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“…However, for any ionizable residues that are in their neutral state this approximation can introduce large errors. For example, the Cβ chemical shifts of Asp and His change by 3.0 and 2.4 ppm due to protonation state changes in small peptides, while the N-chemical shifts change by 1.5 and 1.8 ppm (Platzer et al, 2014). This issue will be addressed in future studies.…”

Section: Backbone Scansmentioning

confidence: 96%

ProCS15: A DFT-based chemical shift predictor for backbone and C β atoms in proteins

Larsen

Bratholm

Christensen

et al. 2015

Preprint

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We present ProCS15: A program that computes the isotropic chemical shielding values of backbone and Cβ atoms given a protein structure in less than a second. ProCS15 is based on around 2.35 million OPBE/6-31G(d,p)//PM6 calculations on tripeptides and small structural models of hydrogen-bonding. The ProCS15-predicted chemical shielding values are compared to experimentally measured chemical shifts for Ubiquitin and the third IgG-binding domain of Protein G through linear regression and yield RMSD values below 2.2, 0.7, and 4.8 ppm for carbon, hydrogen, and nitrogen atoms respectively. These RMSD values are very similar to corresponding RMSD values computed using OPBE/6-31G(d,p) for the entire structure for each protein. The maximum RMSD values can be reduced by using NMR-derived structural ensembles of Ubiquitin. For example, for the largest ensemble the largest RMSD values are 1.7, 0.5, and 3.5 ppm for carbon, hydrogen, and nitrogen. The corresponding RMSD values predicted by several empirical chemical shift predictors range between 0.7 -1.1, 0.2 -0.4, and 1.8 -2.8 ppm for carbon, hydrogen, and nitrogen atoms, respectively.

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“…Thus the pKa of Glu31 was not measured. The pKa values of Asp, Glu, and His in the absence of interactions (e.g., in the GlyXGly tripeptide) are 3.9, 4.3 and 6.4, respectively18. Thus Asp50 and Asp109 have significantly upshifted pKa values, while E47, E92, E97, E121, His86, and His119 have significantly downshifted pKa values.…”

Section: Resultsmentioning

confidence: 98%

“…The protonation state of sidechains of acidic and basic residues depends on their pKa values and the environmental pH. The standard pKa values of Asp, Glu, His, Arg and Lys are 3.9, 4.3, 6.4, 13.9, and 10.3, respectively, when their sidechains are isolated without any interactions with their proximal residues18. Due to charge-charge interactions, hydrogen bonding, and desolvation effects, the pKa values of the acidic and basic residues of a protein often deviate from the standard values22.…”

Section: Discussionmentioning

confidence: 99%

Structural Basis for pH-mediated Regulation of F-actin Severing by Gelsolin Domain 1

Fan

Goh

Ding

et al. 2017

Sci Rep

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Six-domain gelsolin regulates actin structural dynamics through its abilities to sever, cap and uncap F-actin. These activities are modulated by various cellular parameters like Ca2+ and pH. Until now, only the molecular activation mechanism of gelsolin by Ca2+ has been understood relatively well. The fragment comprising the first domain and six residues from the linker region into the second domain has been shown to be similar to the full-length protein in F-actin severing activity in the absence of Ca2+ at pH 5. To understand how this gelsolin fragment is activated for F-actin severing by lowering pH, we solved its NMR structures at both pH 7.3 and 5 in the absence of Ca2+ and measured the pKa values of acidic amino acid residues and histidine residues. The overall structure and dynamics of the fragment are not affected significantly by pH. Nevertheless, local structural changes caused by protonation of His29 and Asp109 result in the activation on lowering the pH, and protonation of His151 directly effects filament binding since it resides in the gelsolin/actin interface. Mutagenesis studies support that His29, Asp109 and His151 play important roles in the pH-dependent severing activity of the gelsolin fragment.

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pH-dependent random coil 1H, 13C, and 15N chemical shifts of the ionizable amino acids: a guide for protein pK a measurements

Cited by 196 publications

References 214 publications

Ubiquitin S65 phosphorylation engenders a pH-sensitive conformational switch

Ubiquitin S65 phosphorylation engenders a pH-sensitive conformational switch

ProCS15: A DFT-based chemical shift predictor for backbone and C β atoms in proteins

Structural Basis for pH-mediated Regulation of F-actin Severing by Gelsolin Domain 1

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