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Using ¹⁵N-ammonium to characterise and map potassium binding sites in proteins by NMR spectroscopy.

Werbeck ND, Kirkpatrick J, Reinstein J, Hansen DF - Chembiochem (2014)

Bottom Line: Here, we demonstrate the use of NMR spectroscopy to characterise binding of ammonium ions to two different enzymes: human histone deacetylase 8 (HDAC8), which is activated allosterically by potassium, and the bacterial Hsp70 homologue DnaK, for which potassium is an integral part of the active site.Ammonium activates both enzymes in a similar way to potassium, thus supporting this non-invasive approach.Furthermore, we present an approach to map the observed binding site onto the structure of HDAC8.

View Article: PubMed Central - PubMed

Affiliation: Institute of Structural and Molecular Biology, Division of Biosciences, University College London, Gower Street, London, WC1E 6BT (UK). d.hansen@ucl.ac.uk.

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A) 15N-edited 1D proton spectrum of the buffer (25 mm Tris⋅HCl pH 8.0, 200 mm15NH4Cl, 0.5 mm TCEP) and direct 15N-detected 1D spectrum (inset). B) As above but after addition of ∼100 μm [14N]HDAC8 to the sample. C) 1H,15N HSQC spectrum of the sample shown in B). D) Titration of 15NH4+ (○) with the observed binding site in HDAC8 (using 15N-edited 1D proton spectra). The intensities were obtained by integration of the ammonium peak, and errors were estimated by a combination of integrating multiple noise regions and by taking into account the uncertainties in calculating the relative protein concentration. At 100 mm15NH4+, addition of 100 mm KCl (▵) led to approximately 50 % reduction of the signal, thus indicating that K+ and NH4+ bind to the same site with similar affinities. A hyperbolic binding equation Y=A×X/(KD+X) (—) was fitted to the normalised peak intensities to yield KD=(13±8) mm.
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fig02: A) 15N-edited 1D proton spectrum of the buffer (25 mm Tris⋅HCl pH 8.0, 200 mm15NH4Cl, 0.5 mm TCEP) and direct 15N-detected 1D spectrum (inset). B) As above but after addition of ∼100 μm [14N]HDAC8 to the sample. C) 1H,15N HSQC spectrum of the sample shown in B). D) Titration of 15NH4+ (○) with the observed binding site in HDAC8 (using 15N-edited 1D proton spectra). The intensities were obtained by integration of the ammonium peak, and errors were estimated by a combination of integrating multiple noise regions and by taking into account the uncertainties in calculating the relative protein concentration. At 100 mm15NH4+, addition of 100 mm KCl (▵) led to approximately 50 % reduction of the signal, thus indicating that K+ and NH4+ bind to the same site with similar affinities. A hyperbolic binding equation Y=A×X/(KD+X) (—) was fitted to the normalised peak intensities to yield KD=(13±8) mm.

Mentions: Next, we tested whether it was possible to observe binding of 15NH4+ to HDAC8 by NMR. As expected, bulk 15NH4+ was not observed in 15N-edited, proton-detected 1D NMR spectra in buffer containing 200 mm15NH4+ at pH 8.0 (Figure 2 A) because of rapid exchange of the ammonium protons with the bulk solvent. The 15N chemical shift of the free ammonium was therefore determined by direct 15N-detection to be 20.5 ppm (Figure 2 A, inset). Addition of [14N]HDAC8, however, resulted in a distinct peak in the 15N-edited experiment, indicative of HDAC8-bound 15NH4+ with a proton chemical shift of 7.1 ppm (Figure 2 B). A 15N chemical shift of 25.5 ppm was subsequently determined by recording a 2D 1H,15N HSQC spectrum (Figure 2 C).


Using ¹⁵N-ammonium to characterise and map potassium binding sites in proteins by NMR spectroscopy.

Werbeck ND, Kirkpatrick J, Reinstein J, Hansen DF - Chembiochem (2014)

A) 15N-edited 1D proton spectrum of the buffer (25 mm Tris⋅HCl pH 8.0, 200 mm15NH4Cl, 0.5 mm TCEP) and direct 15N-detected 1D spectrum (inset). B) As above but after addition of ∼100 μm [14N]HDAC8 to the sample. C) 1H,15N HSQC spectrum of the sample shown in B). D) Titration of 15NH4+ (○) with the observed binding site in HDAC8 (using 15N-edited 1D proton spectra). The intensities were obtained by integration of the ammonium peak, and errors were estimated by a combination of integrating multiple noise regions and by taking into account the uncertainties in calculating the relative protein concentration. At 100 mm15NH4+, addition of 100 mm KCl (▵) led to approximately 50 % reduction of the signal, thus indicating that K+ and NH4+ bind to the same site with similar affinities. A hyperbolic binding equation Y=A×X/(KD+X) (—) was fitted to the normalised peak intensities to yield KD=(13±8) mm.
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Related In: Results  -  Collection

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fig02: A) 15N-edited 1D proton spectrum of the buffer (25 mm Tris⋅HCl pH 8.0, 200 mm15NH4Cl, 0.5 mm TCEP) and direct 15N-detected 1D spectrum (inset). B) As above but after addition of ∼100 μm [14N]HDAC8 to the sample. C) 1H,15N HSQC spectrum of the sample shown in B). D) Titration of 15NH4+ (○) with the observed binding site in HDAC8 (using 15N-edited 1D proton spectra). The intensities were obtained by integration of the ammonium peak, and errors were estimated by a combination of integrating multiple noise regions and by taking into account the uncertainties in calculating the relative protein concentration. At 100 mm15NH4+, addition of 100 mm KCl (▵) led to approximately 50 % reduction of the signal, thus indicating that K+ and NH4+ bind to the same site with similar affinities. A hyperbolic binding equation Y=A×X/(KD+X) (—) was fitted to the normalised peak intensities to yield KD=(13±8) mm.
Mentions: Next, we tested whether it was possible to observe binding of 15NH4+ to HDAC8 by NMR. As expected, bulk 15NH4+ was not observed in 15N-edited, proton-detected 1D NMR spectra in buffer containing 200 mm15NH4+ at pH 8.0 (Figure 2 A) because of rapid exchange of the ammonium protons with the bulk solvent. The 15N chemical shift of the free ammonium was therefore determined by direct 15N-detection to be 20.5 ppm (Figure 2 A, inset). Addition of [14N]HDAC8, however, resulted in a distinct peak in the 15N-edited experiment, indicative of HDAC8-bound 15NH4+ with a proton chemical shift of 7.1 ppm (Figure 2 B). A 15N chemical shift of 25.5 ppm was subsequently determined by recording a 2D 1H,15N HSQC spectrum (Figure 2 C).

Bottom Line: Here, we demonstrate the use of NMR spectroscopy to characterise binding of ammonium ions to two different enzymes: human histone deacetylase 8 (HDAC8), which is activated allosterically by potassium, and the bacterial Hsp70 homologue DnaK, for which potassium is an integral part of the active site.Ammonium activates both enzymes in a similar way to potassium, thus supporting this non-invasive approach.Furthermore, we present an approach to map the observed binding site onto the structure of HDAC8.

View Article: PubMed Central - PubMed

Affiliation: Institute of Structural and Molecular Biology, Division of Biosciences, University College London, Gower Street, London, WC1E 6BT (UK). d.hansen@ucl.ac.uk.

Show MeSH