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, Nicolas D.; Kirkpatrick, John; Hansen, D. Flemming

doi:10.1002/anie.201209385

Cited by 38 publications

(39 citation statements)

References 29 publications

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“…Secondly, the CLEANEX‐PM experiment assumes the slow tumbling limit in order to achieve a cancelation of intramolecular NOE and ROE contributions. The effective correlation time of arginine side‐chains is often so that the slow‐tumbling limit is not applicable, as for the backbone of intrinsically disordered proteins . Thirdly, the use of 13 C detection neatly sidesteps the need to suppress the solvent during acquisition, which can also affect cross‐peaks of interest.…”

Section: Resultsmentioning

confidence: 99%

Arginine Side‐Chain Hydrogen Exchange: Quantifying Arginine Side‐Chain Interactions in Solution

Mackenzie

Hansen

2018

ChemPhysChem

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The rate with which labile backbone hydrogen atoms in proteins exchange with the solvent has long been used to probe protein interactions in aqueous solutions. Arginine, an essential amino acid found in many interaction interfaces, is capable of an impressive range of interactions via its guanidinium group. The hydrogen exchange rate of the guanidinium hydrogens therefore becomes an important measure to quantify side-chain interactions. Herein we present an NMR method to quantify the hydrogen exchange rates of arginine side-chain H protons and thus present a method to gauge the strength of arginine side-chain interactions. The method employs C-detection and the one-bond deuterium isotope shift observed for N to generate two exchanging species in H O/ H O mixtures. An application to the protein T4 Lysozyme is shown, where protection factors calculated from the obtained exchange rates correlate well with the interactions observed in the crystal structure. The methodology presented provides an important step towards characterising interactions of arginine side-chains in enzymes, in phase separation, and in protein interaction interfaces in general.

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Section: Resultsmentioning

confidence: 99%

Arginine Side‐Chain Hydrogen Exchange: Quantifying Arginine Side‐Chain Interactions in Solution

Mackenzie

Hansen

2018

ChemPhysChem

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“…Although caution must be exercised in inferring charge states from chemical shifts, all 13 had 15 N ε , 13 C ζ , and 15 N η signals highly indicative of a fully protonated guanidinium sidechain (e.g., 84.8 ppm, 159.5 ppm, and 75.0 and ~ 71 ppm, respectively, for Arg145) . Furthermore, the 13 C and 15 N NMR signals of corresponding arginines in the T26H–T4L* mutant and in the wild‐type protein are very similar . Of these, several, including Arg145, in T4L have been unambiguously demonstrated to be fully protonated at pH 5.5, and hence positively charged, on the basis of 1 H η – 15 N η scalar coupling patterns …”

Section: Resultsmentioning

confidence: 99%

“…Histidine imidazole signals were readily identified in conventional and constant time 13 C‐HSQC spectra . Arginine 15 N ε signals were assigned from an H ε N ε C δ spectrum, recorded using a modified HNCA experiment, 13 C ζ signals from an H ε N ε C ζ spectrum, recorded using a modified HNCO experiment, and 15 N η signals from a cross polarization 13 C‐detected N ε/η ‐C ζ correlation experiment . The modifications involved setting the transmitter frequencies for 15 N ε (84.5 ppm), 13 C δ (43.5 ppm), and 13 C ζ (160 ppm), and lengthening the 1 H ε – 15 N ε de‐phasing/re‐phasing delays to 2.8 ms along with a 1.67 msec 15 N ε ‐selective 180° pulse.…”

Section: Methodsmentioning

confidence: 99%

The pK_a values of the catalytic residues in the retaining glycoside hydrolase T26H mutant of T4 lysozyme

et al. 2019

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T4 phage lysozyme (T4L) is an enzyme that cleaves bacterial cell wall peptidoglycan. Remarkably, the single substitution of the active site Thr26 to a His (T26H) converts T4L from an inverting to a retaining glycoside hydrolase with transglycosylase activity. It has been proposed that T26H–T4L follows a double displacement mechanism with His26 serving as a nucleophile to form a covalent glycosyl‐enzyme intermediate (Kuroki et al., PNAS 1999; 96:8949–8954). To gain further insights into this or alternative mechanisms, we used NMR spectroscopy to measure the acid dissociation constants (pKa values) and/or define the ionization states of the Asp, Glu, His, and Arg residues in the T4L mutant. Most notably, the pKa value of the putative nucleophile His26 is 6.8 ± 0.1, whereas that of the general acid Glu11 is 4.7 ± 0.1. If the proposed mechanism holds true, then T26H–T4L follows a reverse protonation pathway in which only a minor population of the free enzyme is in its catalytically competent ionization state with His26 deprotonated and Glu11 protonated. Our studies also confirm that all arginines in T26H–T4L, including the active site Arg145, are positively charged under neutral pH conditions. Brief statement The replacement of a single amino acid changes T4 lysozyme from an inverting to a retaining glycoside hydrolase. Using NMR spectroscopy, we measured the pKa values of the ionizable residues in the active site of this mutant enzyme. Along with previously reported data, these results provide important constraints for understanding the catalytic mechanisms by which the wild‐type and mutant form of T4 lysozyme cleave bacterial peptidoglycan.

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“…The NMR chemical shifts of the arginine head‐group 15 N ϵ , 15 N η , and 13 C ζ nuclei are sensitive to the charge state, and a dispersion of the chemical shift in pH titration experiments can be used to determine the side chain p K a value. The 15 N ϵ and 13 C ζ chemical shifts can be obtained in 2D HD(CD)NE, HD(CDNE)CZ, and 15 N ϵ – 13 C ζ correlation spectra . For PYP we attempted pH titration experiments and followed the 13 C ζ and 15 N ϵ resonances of R52.…”

Section: Figurementioning

confidence: 99%

“…In summary, we show that the charge states of individual arginine side chains in proteins are accessible by the 13 C‐detected experiments presented herein. The methodology demonstrated may be useful for large folded proteins as well because side‐chain and backbone motions are often uncoupled …”

Section: Figurementioning

confidence: 99%

Unambiguous Determination of Protein Arginine Ionization States in Solution by NMR Spectroscopy

et al. 2016

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Because arginine residues in proteins are expected to be in their protonated form almost without exception, reports demonstrating that aprotein arginine residue is charge-neutral are rare and potentially controversial. Herein, we present a 13 Cdetected NMR experiment for probing individual arginine residues in proteins notwithstanding the presence of chemical and conformational exchange effects.I nt he experiment, the 15 N h and 15 N e chemical shifts of an arginine head group are correlated with that of the directly attached 13 C z .Inthe resulting spectrum, the number of protons in the arginine head group can be obtained directly from the 15 N-1 Hs calar coupling splitting pattern. We applied this method to unambiguously determine the ionization state of the R52 side chain in the photoactive yellow protein from Halorhodospira halophila. Although only three H h atoms were previously identified by neutron crystallography,w es howt hat R52 is predominantly protonated in solution.Supportinginformation, includinge xperimental details, and the ORCID identification number(s) for the author(s) of this article can be found under: http://dx.

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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

Cited by 38 publications

References 29 publications

Arginine Side‐Chain Hydrogen Exchange: Quantifying Arginine Side‐Chain Interactions in Solution

Arginine Side‐Chain Hydrogen Exchange: Quantifying Arginine Side‐Chain Interactions in Solution

The pK_a values of the catalytic residues in the retaining glycoside hydrolase T26H mutant of T4 lysozyme

Unambiguous Determination of Protein Arginine Ionization States in Solution by NMR Spectroscopy

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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

Cited by 38 publications

References 29 publications

Arginine Side‐Chain Hydrogen Exchange: Quantifying Arginine Side‐Chain Interactions in Solution

Arginine Side‐Chain Hydrogen Exchange: Quantifying Arginine Side‐Chain Interactions in Solution

The pKa values of the catalytic residues in the retaining glycoside hydrolase T26H mutant of T4 lysozyme

Unambiguous Determination of Protein Arginine Ionization States in Solution by NMR Spectroscopy

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The pK_a values of the catalytic residues in the retaining glycoside hydrolase T26H mutant of T4 lysozyme