Additions and Corrections - Nuclear Magnetic Resonance Investigation of <sup>15</sup>N-Labeled Histidine in Aqueous Solutions.

Blomberg, F.; Mäurer, W; Rüterjans, H.

doi:10.1021/ja00477a607

Cited by 7 publications

(8 citation statements)

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“…The imidazole ring nitrogens of this residue have I5N chemical shifts at 188.0 and 174.4 ppm, as shown in Figure 1; these have been tentatively assigned to N6' and Nf2, respectively, using HMQC and HMBC spectra . The cross peak between H" and N61 is significantly weaker than between this proton and Nf2 (Blomberg et al, 1977). The pK, of His31 is approximately 9.1 (Anderson et al, 1990), and both the proton and nitrogen chemical shifts of the imidazole ring indicated that the histidine is in its acidic form at the pH of these measurements.…”

Section: Assignment Of the Proton Resonances From The Aromatic Side Cmentioning

confidence: 76%

Assignment of the backbone proton and nitrogen-15 NMR resonances of bacteriophage T4 lysozyme

McIntosh

Wand²,

Lowry³

et al. 1990

Biochemistry

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The proton and nitrogen (15NH-H alpha-H beta) resonances of bacteriophage T4 lysozyme were assigned by 15N-aided 1H NMR. The assignments were directed from the backbone amide 1H-15N nuclei, with the heteronuclear single-multiple-quantum coherence (HSMQC) spectrum of uniformly 15N enriched protein serving as the master template for this work. The main-chain amide 1H-15N resonances and H alpha resonances were resolved and classified into 18 amino acid types by using HMQC and 15N-edited COSY measurements, respectively, of T4 lysozymes selectively enriched with one or more of alpha-15N-labeled Ala, Arg, Asn, Asp, Gly, Gln, Glu, Ile, Leu, Lys, Met, Phe, Ser, Thr, Trp, Tyr, or Val. The heteronuclear spectra were complemented by proton DQF-COSY and TOCSY spectra of unlabeled protein in H2O and D2O buffers, from which the H beta resonances of many residues were identified. The NOE cross peaks to almost every amide proton were resolved in 15N-edited NOESY spectra of the selectively 15N enriched protein samples. Residue specific assignments were determined by using NOE connectivities between protons in the 15NH-H alpha-H beta spin systems of known amino acid type. Additional assignments of the aromatic proton resonances were obtained from 1H NMR spectra of unlabeled and selectively deuterated protein samples. The secondary structure of T4 lysozyme indicated from a qualitative analysis of the NOESY data is consistent with the crystallographic model of the protein.

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Section: Assignment Of the Proton Resonances From The Aromatic Side Cmentioning

confidence: 76%

Assignment of the backbone proton and nitrogen-15 NMR resonances of bacteriophage T4 lysozyme

McIntosh

Wand²,

Lowry³

et al. 1990

Biochemistry

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“…Since the semiquinone resonance is nearer to the reduced than to the oxidized resonance, the phosphate binding conformation of the semiquinone more closely resembles that of reduced flavodoxin than that of oxidized flavodoxin. (Blomberg et al, 1977) because hydrogen bonding to these nitrogens may significantly alter their chemical shifts, as has been observed for the catalytic triad histidine of cy-lytic protease (Bachovchin & Roberts, 1978). Other interesting regions in both spectra are as follows: 105-135 ppm, amide backbone nitrogens and glutamine and asparagine side-chain nitrogens; 84-88 and 72-77 ppm, arginine 6-and q-side-chain nitrogens, respectively; and 3 1-36 ppm, lysine e-amino nitrogens and N-terminal nitrogen.…”

Section: P N M R On Reduced and Oxidized Flavodoxinmentioning

confidence: 91%

Flavodoxin from Anabaena 7120: uniform nitrogen-15 enrichment and hydrogen-1, nitrogen-15, and phosphorus-31 NMR investigations of the flavin mononucleotide binding site in the reduced and oxidized states

Stockman

Westler²,

Mooberry³

et al. 1988

Biochemistry

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Interactions between flavin mononucleotide (FMN) and apoprotein have been investigated in the reduced and oxidized states of the flavodoxin isolated from Anabaena 7120 (Mr approximately 21,000). 1H, 15N, and 31P NMR have been used to characterize the FMN-protein interactions in both redox states. These are compared with those seen in other flavodoxins. Uniformly enriched [15N]flavodoxin (greater than 95% isotopic purity) was isolated from Anabaena 7120 grown on K15NO3 as the sole nitrogen source. 15N insensitive nucleus enhanced by polarization transfer (INEPT) and nuclear Overhauser effect (NOE) studies of this sample provided information regarding protein structure and dynamics. A 1H-detected 15N experiment allowed the correlation of nitrogen resonances to those of their attached protons. Over 90% of the expected N-H cross peaks could be resolved in this experiment.

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“…The observed average nitrogen chemical shifts δ obs of 1-3 can then be expressed as a function of pH in terms of the Henderson-Hasselbalch equation, 33,34 adapted for NMR spectroscopic measurements 35 in the fast proton-transfer regime Here, pK a (ifi+1) ) -log K a (ifi+1), where K a (ifi+1) represents the equilibrium constant of the formation of protonation state i + 1. δ N represents the 15 N chemical shift in state i + 1, which is the limiting value for the deprotonated nitrogen atom. δ NH represents the 15 N chemical shift in state i, which is the limiting value of the protonated nitrogen atom.…”

Section: Methodsmentioning

confidence: 99%

¹⁵N Nuclear Magnetic Resonance Studies of Acid−Base Properties of Pyridoxal-5‘-Phosphate Aldimines in Aqueous Solution

et al. 2007

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By use of 15N NMR spectroscopy, we have measured the pKa values of the aldimines 15N-(pyridoxyl-5'-phosphate-idine)-methylamine (2a), N-(pyridoxyl-5'-phosphate-15N-idine)-methylamine (2b), and 15N-(pyridoxyl-idine)-methylamine (3). These aldimines model the cofactor pyridoxal-5'-phosphate (PLP, 1) in a variety of PLP-dependent enzymes. The acid-base properties of the aldimines differ substantially from those of the free cofactor in the aldehyde form 1a or in the hydrated form 1b, which were also investigated using 15N NMR for comparison. All compounds contain three protonation sites, the pyridine ring, the phenol group, and the side chain phosphate (1, 2) or hydroxyl group (3). In agreement with the literature, 1a exhibits one of several pKas at 2.9 and 1b at 4.2. The 15N chemical shifts indicate that the corresponding deprotonation occurs partially in the pyridine and partially in the phenolic site, which compete for the remaining proton. The equilibrium constant of this ring-phenolate tautomerism was measured to be 0.40 for 1a and 0.06 for 1b. The tautomerism is essentially unaltered above pH 6.1, where the phosphate group is deprotonated to the dianion. This means that the pyridine ring is more basic than the phenolate group. Pyridine nitrogen deprotonation occurs at 8.2 for 1a and at 8.7 for 1b. By contrast, above pH 4 the phosphate site of 2 is deprotonated, while the pyridine ring pKa is 5.8. The Schiff base nitrogen does not deprotonate below pH 11.4. When the phosphate group is removed, the pKa of the Schiff base nitrogen decreases to 10.5. The phenol site cannot compete for the proton of the Schiff base nitrogen and is present in the entire pH range as a phenolate, preferentially hydrogen bonded to the solvent. The intrinsic 15N chemical shifts provide information about the hydrogen bond structures of the protonated and unprotonated species involved. Evidence is presented that the intramolecular OHN hydrogen bond of PLP aldimines is broken in aqueous solution. The coupling between the inter- and intramolecular OHN hydrogen bonds is also lost in this environment. The pyridine ring of the PLP aldimines is not protonated in aqueous solution near neutral pH. The basicity of the aldimine nitrogens would be even lower without the doubly negatively charged phosphate group. Protonation of both the Schiff base and pyridine nitrogens has been discussed as a prerequisite for catalytic activity, and the implications of the present findings for PLP catalysis are discussed.

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Additions and Corrections - Nuclear Magnetic Resonance Investigation of ¹⁵N-Labeled Histidine in Aqueous Solutions.

Cited by 7 publications

References 0 publications

Assignment of the backbone proton and nitrogen-15 NMR resonances of bacteriophage T4 lysozyme

Assignment of the backbone proton and nitrogen-15 NMR resonances of bacteriophage T4 lysozyme

Flavodoxin from Anabaena 7120: uniform nitrogen-15 enrichment and hydrogen-1, nitrogen-15, and phosphorus-31 NMR investigations of the flavin mononucleotide binding site in the reduced and oxidized states

¹⁵N Nuclear Magnetic Resonance Studies of Acid−Base Properties of Pyridoxal-5‘-Phosphate Aldimines in Aqueous Solution

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Additions and Corrections - Nuclear Magnetic Resonance Investigation of 15N-Labeled Histidine in Aqueous Solutions.

Cited by 7 publications

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Assignment of the backbone proton and nitrogen-15 NMR resonances of bacteriophage T4 lysozyme

Assignment of the backbone proton and nitrogen-15 NMR resonances of bacteriophage T4 lysozyme

Flavodoxin from Anabaena 7120: uniform nitrogen-15 enrichment and hydrogen-1, nitrogen-15, and phosphorus-31 NMR investigations of the flavin mononucleotide binding site in the reduced and oxidized states

15N Nuclear Magnetic Resonance Studies of Acid−Base Properties of Pyridoxal-5‘-Phosphate Aldimines in Aqueous Solution

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Additions and Corrections - Nuclear Magnetic Resonance Investigation of ¹⁵N-Labeled Histidine in Aqueous Solutions.

¹⁵N Nuclear Magnetic Resonance Studies of Acid−Base Properties of Pyridoxal-5‘-Phosphate Aldimines in Aqueous Solution