The pH-dependence of amide chemical shift of Asp/Glu reflects its pKa in intrinsically disordered proteins with only local interactions

Pujato, Mario; Navarro, Abel E.; Versace, Rodney; Mancusso, Romina; Ghose, Ranajeet; Tasayco, María Luisa

doi:10.1016/j.bbapap.2006.04.014

Cited by 17 publications

(11 citation statements)

References 39 publications

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“…While we could obtain accurate data for the Cγ resonance of Glu114 from the C(CO)NH experiments, the titration of Glu61 was more accurately determined from the 15 N resonance in 2D 1 H‐ 15 N HSQC experiments. Consistent with previous results on unfolded proteins,45 we found that the 15 N shifts of ionizable residues in αS are dominated by the protonation of the residue's own side‐chain.…”

Section: Resultssupporting

confidence: 92%

NMR determination of pK_a values in α‐synuclein

et al. 2010

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The intrinsically unfolded protein a-synuclein has an N-terminal domain with seven imperfect KTKEGV sequence repeats and a C-terminal domain with a large proportion of acidic residues. We characterized pK a values for all 26 sites in the protein that ionize below pH 7 using 2D 1 H-15 N HSQC and 3D C(CO)NH NMR experiments. The N-terminal domain shows systematically lowered pK a values, suggesting weak electrostatic interactions between acidic and basic residues in the KTKEGV repeats. By contrast, the C-terminal domain shows elevated pK a values due to electrostatic repulsion between like charges. The effects are smaller but persist at physiological salt concentrations. For a-synuclein in the membrane-like environment of sodium dodecylsulfate (SDS) micelles, we characterized the pK a of His50, a residue of particular interest since it is flanked within one turn of the a-helix structure by the Parkinson's disease-linked mutants E46K and A53T. The pK a of His50 is raised by 1.4 pH units in the micelle-bound state. Titrations of His50 in the micelle-bound states of the E46K and A53T mutants show that the pK a shift is primarily due to interactions between the histidine and the sulfate groups of SDS, with electrostatic interactions between His50 and Glu46 playing a much smaller role. Our results indicate that the pK a values of uncomplexed a-synuclein differ significantly from random coil model peptides even though the protein is intrinsically unfolded. Due to the long-range nature of electrostatic interactions, charged residues in the a-synuclein sequence may help nucleate the folding of the protein into an a-helical structure and confer protection from misfolding.

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Section: Resultssupporting

confidence: 92%

NMR determination of pK_a values in α‐synuclein

et al. 2010

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“…The pH dependence of the C β chemical shifts ranged from ∼39 to ∼41 ppm [Fig. (A), Table ]; these values were similar to those observed in the protonated and deprotonated species of other disordered proteins or protein fragments . In all cases, the Hill coefficients, n , for the titrations of the C β resonances were ∼1.0 (Table ).…”

Section: Resultssupporting

confidence: 55%

Intrinsically disordered inhibitor of glutamine synthetase is a functional protein with random‐coil‐like pK_a values

et al. 2017

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The sequential action of glutamine synthetase (GS) and glutamate synthase (GOGAT) in cyanobacteria allows the incorporation of ammonium into carbon skeletons. In the cyanobacterium Synechocystis sp. PCC 6803, the activity of GS is modulated by the interaction with proteins, which include a 65-residue-long intrinsically disordered protein (IDP), the inactivating factor IF7. This interaction is regulated by the presence of charged residues in both IF7 and GS. To understand how charged amino acids can affect the binding of an IDP with its target and to provide clues on electrostatic interactions in disordered states of proteins, we measured the pK values of all IF7 acidic groups (Glu32, Glu36, Glu38, Asp40, Asp58, and Ser65, the backbone C-terminus) at 100 mM NaCl concentration, by using NMR spectroscopy. We also obtained solution structures of IF7 through molecular dynamics simulation, validated them on the basis of previous experiments, and used them to obtain theoretical estimates of the pK values. Titration values for the two Asp and three Glu residues of IF7 were similar to those reported for random-coil models, suggesting the lack of electrostatic interactions around these residues. Furthermore, our results suggest the presence of helical structure at the N-terminus of the protein and of conformational changes at acidic pH values. The overall experimental and in silico findings suggest that local interactions and conformational equilibria do not play a role in determining the electrostatic features of the acidic residues of IF7.

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“…In contrast, the nonacidic residues display small chemical shift changes, of 0.4 ppm or less for most residues and one of the exceptions is Q55 which is affected by titration of the C‐terminus (Supporting Information). This strongly suggests that the amide 15 N chemical shift changes of the acidic residues are due to protonation of their side chain carboxylates and indicates that 15 N shifts can be used as a reliable p K a predictor 33. Each acidic residue shows a sigmoidal titration curve with no sign of a multiphasic transition (Fig.…”

Section: Resultsmentioning

confidence: 84%

“…Complicated multiphase 1 H and 15 N titration curves were observed for several sites indicating that, in these cases, they do not report simply on the ionization state of their side chains. In other systems 15 N titration curves have been observed to correlate very closely with side chain 13 C titration curves 33. The six acidic residues of the V3AI4A‐NTL9 DSE exhibit large 15 N chemical shift changes during the titration, ranging from 0.81 ppm to 1.66 ppm.…”

Section: Resultsmentioning

confidence: 85%

Analysis of electrostatic interactions in the denatured state ensemble of the N‐terminal domain of L9 under native conditions

Meng

Raleigh

2011

Proteins

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The pH dependence of protein stability is defined by the difference in the number of protons bound to the folded state and to the denatured state ensemble (DSE) as a function of pH. In many cases, the protonation behavior can be described as the sum of a set of independently titrating residues; in this case, the pH dependence of stability reflects differences in folded and DSE pK(a)'s. pH dependent stability studies have shown that there are energetically important interactions involving charged residues in the DSE of the N-terminal domain of L9 (NTL9), which affect significantly the stability of the protein. The DSE of wild type NTL9 cannot be directly characterized under native conditions because of its high stability. A destabilized double mutant of NTL9, V3AI4A, significantly populates the folded state and the DSE in the absence of denaturant. The two states are in slow exchange on the nuclear magnetic resonance time scale, and diffusion measurements indicate that the DSE is compact. The DSE pK(a)'s of all of the acidic residues were directly determined. The DSE pK(a) of Asp8 and Asp23 are depressed relative to model compounds values. Use of the mutant DSE pK(a)'s together with known native state pK(a)'s leads to a significantly improved agreement between the measured pH dependent stability and that predicted by the Tanford-Wyman linkage relationship. An analysis of the literature suggests that DSE interactions involving charged residues are relatively common and should be considered in discussions of protein stability.

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The pH-dependence of amide chemical shift of Asp/Glu reflects its pKa in intrinsically disordered proteins with only local interactions

Cited by 17 publications

References 39 publications

NMR determination of pK_a values in α‐synuclein

NMR determination of pK_a values in α‐synuclein

Intrinsically disordered inhibitor of glutamine synthetase is a functional protein with random‐coil‐like pK_a values

Analysis of electrostatic interactions in the denatured state ensemble of the N‐terminal domain of L9 under native conditions

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The pH-dependence of amide chemical shift of Asp/Glu reflects its pKa in intrinsically disordered proteins with only local interactions

Cited by 17 publications

References 39 publications

NMR determination of pKa values in α‐synuclein

NMR determination of pKa values in α‐synuclein

Intrinsically disordered inhibitor of glutamine synthetase is a functional protein with random‐coil‐like pKa values

Analysis of electrostatic interactions in the denatured state ensemble of the N‐terminal domain of L9 under native conditions

Contact Info

Product

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NMR determination of pK_a values in α‐synuclein

NMR determination of pK_a values in α‐synuclein

Intrinsically disordered inhibitor of glutamine synthetase is a functional protein with random‐coil‐like pK_a values