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Probing arginine side-chains and their dynamics with carbon-detected NMR spectroscopy: application to the 42 kDa human histone deacetylase 8 at high pH.

Werbeck ND, Kirkpatrick J, Hansen DF - Angew. Chem. Int. Ed. Engl. (2013)

View Article: PubMed Central - PubMed

Affiliation: Institute of Structural and Molecular Biology, University College London, Gower Street, WC1E 6BT, London, UK.

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In contrast to other charged amino acid side-chains the charge of arginine side-chains is relatively unaffected by the surrounding environment... Spin relaxation rates were obtained from a series of Cζ–Nε spectra and order parameters were subsequently calculated for the arginine side-chains of T4L L99A... Figure 2 d shows a good agreement when these order parameters are compared to the corresponding order parameters, SHN, obtained using proton-detected experiments... Thus, the Hε–Nε spectrum is not applicable to probe the arginine side-chains of HDAC8, as shown in Figure 3 a, and these side-chains must be probed independently of the Hε spin... Figure 3 a and 3 b show a Hε–Nε and a Cζ–Nε spectrum, respectively, of HDAC8 at pH 8.2 and clearly demonstrate that under these conditions the Cζ–Nε spectrum reveals many more features than the corresponding Hε–Nε spectrum... Crystal structures of HDAC8 show that the Cζ of R223 is ca. 12 Å from one of the two potassium binding sites, which allows us to investigate the wider consequences on the side-chain packing of K binding... At low concentrations of K the R223 peak is hardly visible, whereas its intensity increases as the concentration of K is increased, Figure 3 d... On the contrary, addition of Na does not lead to the same increase in intensity, thus attesting that the effect observed is due to specific K binding rather than electrostatic stabilization of the protein... In general, the side-chains of amino acids probe a different environment from that of the backbone, and charged side-chains probe a different environment from that of hydrophobic side-chains... A comprehensive list of all experiments including sample details, experimental conditions, and recording parameters is given in the Supporting Information... NMR spectra were processed with NMRpipe and relaxation rates were obtained using FuDA... Order parameters were obtained using model-free approaches as described in the Supporting Information... For the potassium titration of HDAC8 peak volumes were processed as described in the Supporting Information.

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a) Structure of T4L L99A (PDB: 1L90[13]). b) 13Cζ–15Nε spectrum of T4L L99A obtained at 16.4 T (176 MHz 13C frequency) and 298 K with the pulse scheme shown in Figure 1. c) A chemical shift assignment is exemplified with the 13Cζ–15Nε resonance of R80 assigned from the carbon-detected 3D CCNeCz-TOCSY (left; Supporting Information), the 3D CC(CO)NH-TOCSY[14] (right), and the backbone assignment[12] of T4L L99A. d) Comparison of order parameters derived from the carbon-detected experiments, S2CN, with those derived from proton-detected 1Hε–15Nε experiments, S2HN. The order parameters, S2CN, were derived from the R1(2CzNz), R2(2CzNx), and R1(Cz) relaxation rates as described in the Supporting Information. The inset shows examples of decay curves used to determine the relaxation rates: R96: R1(2CzNz)=0.78±0.06 s−1, R14: R1(2CzNz)= 1.32±0.02 s−1. The wide range of order parameters observed for the arginine side-chains of T4L L99A suggests possible motions around the four side-chain dihedral angles, in agreement with results obtained for methyl-bearing side-chains.[15] RMSD=root mean-square deviation.
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fig02: a) Structure of T4L L99A (PDB: 1L90[13]). b) 13Cζ–15Nε spectrum of T4L L99A obtained at 16.4 T (176 MHz 13C frequency) and 298 K with the pulse scheme shown in Figure 1. c) A chemical shift assignment is exemplified with the 13Cζ–15Nε resonance of R80 assigned from the carbon-detected 3D CCNeCz-TOCSY (left; Supporting Information), the 3D CC(CO)NH-TOCSY[14] (right), and the backbone assignment[12] of T4L L99A. d) Comparison of order parameters derived from the carbon-detected experiments, S2CN, with those derived from proton-detected 1Hε–15Nε experiments, S2HN. The order parameters, S2CN, were derived from the R1(2CzNz), R2(2CzNx), and R1(Cz) relaxation rates as described in the Supporting Information. The inset shows examples of decay curves used to determine the relaxation rates: R96: R1(2CzNz)=0.78±0.06 s−1, R14: R1(2CzNz)= 1.32±0.02 s−1. The wide range of order parameters observed for the arginine side-chains of T4L L99A suggests possible motions around the four side-chain dihedral angles, in agreement with results obtained for methyl-bearing side-chains.[15] RMSD=root mean-square deviation.

Mentions: As a first application we probed the arginine side-chains of T4 lysozyme L99A (T4L L99A; 18 kDa) shown in Figure 2 a. T4L L99A is stable at pH 5.5[12] and therefore provides an opportunity for a comparison of the carbon-detected methodology proposed here with the conventional proton-detected methods. The 13Cζ–15Nε spectrum of T4L L99A, Figure 2 b, shows many well-dispersed peaks and a chemical shift dispersion similar to the corresponding 1Hε–15Nε spectrum (Supporting Information). As expected, the signal/noise ratios of the disperse peaks of the 13Cζ–15Nε spectrum are approximately a factor of 30 less than the corresponding peaks of the 1Hε–15Nε spectrum obtained with the same recording time. The proton-detected experiments therefore remain preferable at pH below ca. 6, whereas at higher pH the disadvantage of the longer recording time of the 13Cζ–15Nε spectrum is often outweighed by the fact that the 1Hε–15Nε spectrum provides very limited information.


Probing arginine side-chains and their dynamics with carbon-detected NMR spectroscopy: application to the 42 kDa human histone deacetylase 8 at high pH.

Werbeck ND, Kirkpatrick J, Hansen DF - Angew. Chem. Int. Ed. Engl. (2013)

a) Structure of T4L L99A (PDB: 1L90[13]). b) 13Cζ–15Nε spectrum of T4L L99A obtained at 16.4 T (176 MHz 13C frequency) and 298 K with the pulse scheme shown in Figure 1. c) A chemical shift assignment is exemplified with the 13Cζ–15Nε resonance of R80 assigned from the carbon-detected 3D CCNeCz-TOCSY (left; Supporting Information), the 3D CC(CO)NH-TOCSY[14] (right), and the backbone assignment[12] of T4L L99A. d) Comparison of order parameters derived from the carbon-detected experiments, S2CN, with those derived from proton-detected 1Hε–15Nε experiments, S2HN. The order parameters, S2CN, were derived from the R1(2CzNz), R2(2CzNx), and R1(Cz) relaxation rates as described in the Supporting Information. The inset shows examples of decay curves used to determine the relaxation rates: R96: R1(2CzNz)=0.78±0.06 s−1, R14: R1(2CzNz)= 1.32±0.02 s−1. The wide range of order parameters observed for the arginine side-chains of T4L L99A suggests possible motions around the four side-chain dihedral angles, in agreement with results obtained for methyl-bearing side-chains.[15] RMSD=root mean-square deviation.
© Copyright Policy - open-access
Related In: Results  -  Collection

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Show All Figures
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fig02: a) Structure of T4L L99A (PDB: 1L90[13]). b) 13Cζ–15Nε spectrum of T4L L99A obtained at 16.4 T (176 MHz 13C frequency) and 298 K with the pulse scheme shown in Figure 1. c) A chemical shift assignment is exemplified with the 13Cζ–15Nε resonance of R80 assigned from the carbon-detected 3D CCNeCz-TOCSY (left; Supporting Information), the 3D CC(CO)NH-TOCSY[14] (right), and the backbone assignment[12] of T4L L99A. d) Comparison of order parameters derived from the carbon-detected experiments, S2CN, with those derived from proton-detected 1Hε–15Nε experiments, S2HN. The order parameters, S2CN, were derived from the R1(2CzNz), R2(2CzNx), and R1(Cz) relaxation rates as described in the Supporting Information. The inset shows examples of decay curves used to determine the relaxation rates: R96: R1(2CzNz)=0.78±0.06 s−1, R14: R1(2CzNz)= 1.32±0.02 s−1. The wide range of order parameters observed for the arginine side-chains of T4L L99A suggests possible motions around the four side-chain dihedral angles, in agreement with results obtained for methyl-bearing side-chains.[15] RMSD=root mean-square deviation.
Mentions: As a first application we probed the arginine side-chains of T4 lysozyme L99A (T4L L99A; 18 kDa) shown in Figure 2 a. T4L L99A is stable at pH 5.5[12] and therefore provides an opportunity for a comparison of the carbon-detected methodology proposed here with the conventional proton-detected methods. The 13Cζ–15Nε spectrum of T4L L99A, Figure 2 b, shows many well-dispersed peaks and a chemical shift dispersion similar to the corresponding 1Hε–15Nε spectrum (Supporting Information). As expected, the signal/noise ratios of the disperse peaks of the 13Cζ–15Nε spectrum are approximately a factor of 30 less than the corresponding peaks of the 1Hε–15Nε spectrum obtained with the same recording time. The proton-detected experiments therefore remain preferable at pH below ca. 6, whereas at higher pH the disadvantage of the longer recording time of the 13Cζ–15Nε spectrum is often outweighed by the fact that the 1Hε–15Nε spectrum provides very limited information.

View Article: PubMed Central - PubMed

Affiliation: Institute of Structural and Molecular Biology, University College London, Gower Street, WC1E 6BT, London, UK.

AUTOMATICALLY GENERATED EXCERPT
Please rate it.

In contrast to other charged amino acid side-chains the charge of arginine side-chains is relatively unaffected by the surrounding environment... Spin relaxation rates were obtained from a series of Cζ–Nε spectra and order parameters were subsequently calculated for the arginine side-chains of T4L L99A... Figure 2 d shows a good agreement when these order parameters are compared to the corresponding order parameters, SHN, obtained using proton-detected experiments... Thus, the Hε–Nε spectrum is not applicable to probe the arginine side-chains of HDAC8, as shown in Figure 3 a, and these side-chains must be probed independently of the Hε spin... Figure 3 a and 3 b show a Hε–Nε and a Cζ–Nε spectrum, respectively, of HDAC8 at pH 8.2 and clearly demonstrate that under these conditions the Cζ–Nε spectrum reveals many more features than the corresponding Hε–Nε spectrum... Crystal structures of HDAC8 show that the Cζ of R223 is ca. 12 Å from one of the two potassium binding sites, which allows us to investigate the wider consequences on the side-chain packing of K binding... At low concentrations of K the R223 peak is hardly visible, whereas its intensity increases as the concentration of K is increased, Figure 3 d... On the contrary, addition of Na does not lead to the same increase in intensity, thus attesting that the effect observed is due to specific K binding rather than electrostatic stabilization of the protein... In general, the side-chains of amino acids probe a different environment from that of the backbone, and charged side-chains probe a different environment from that of hydrophobic side-chains... A comprehensive list of all experiments including sample details, experimental conditions, and recording parameters is given in the Supporting Information... NMR spectra were processed with NMRpipe and relaxation rates were obtained using FuDA... Order parameters were obtained using model-free approaches as described in the Supporting Information... For the potassium titration of HDAC8 peak volumes were processed as described in the Supporting Information.

Show MeSH
Related in: MedlinePlus