Nitrogen‐15 n.m.r. studies of <sup>13</sup>C, <sup>15</sup>N labeled arginine

London, Robert E.; Walker, Thomas E.; Whaley, Thomas W.; Matwiyoff, Nicholas A.

doi:10.1002/mrc.1270091011

Cited by 24 publications

(5 citation statements)

References 19 publications

(2 reference statements)

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“…Due to fast rotation about the N ε ‐C ζ and C ζ ‐N η bond, average values are observed for the 1 H η and 15 N η nuclei . Although consistent with earlier studies, the Δ δ values are generally larger than previously published, possibly due to better extrapolation to the high pH plateau chemical shifts …”

Section: Discussionmentioning

confidence: 99%

Arginine: Its pK_a value revisited

et al. 2015

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Using complementary approaches of potentiometry and NMR spectroscopy, we have determined that the equilibrium acid dissociation constant (pK a value) of the arginine guanidinium group is 13.8 6 0.1. This is substantially higher than that of~12 often used in structure-based electrostatics calculations and cited in biochemistry textbooks. The revised intrinsic pK a value helps explains why arginine side chains in proteins are always predominantly charged, even at pH values as great as 10. The high pK a value also reinforces the observation that arginine side chains are invariably protonated under physiological conditions of near neutral pH. This occurs even when the guanidinium moiety is buried in a hydrophobic micro-environment, such as that inside a protein or a lipid membrane, thought to be incompatible with the presence of a charged group.

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Section: Discussionmentioning

confidence: 99%

Arginine: Its pK_a value revisited

et al. 2015

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“…In aqueous solution, the protons attached to these nitrogens undergo base-catalyzed exchange with water protons, and the rates of exchange, 1 /re (where re is the average lifetime between proton transfer), increase with pH. Thus, for the NW|U/ of arginine, the proton-coupled 15N resonance is a sharp triplet with a /N_H of 92 Hz at pH <5, where the exchange is slow on the NMR time scale (re/ » 1), but the resonance broadens and coalesces to a singlet as the exchange rate approaches the order of magnitude of the coupling constant (t,J ~1) at pH 6-7.5 (Blomberg et al, 1976;London et al, 1977;Yavari & Roberts, 1978). For the a-amino nitrogens of alanine and proline, which are less basic, the proton-coupled 15N resonance is a broad singlet at pH <6.0, indicating that the rate is intermediate on the NMR time scale (rj = 1), and the resonance gradually sharpens over pH 6-8 as the rate of exchange becomes fast (tJ «1).…”

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Measurements of cytoplasmic and vacuolar pH in Neurospora using nitrogen-15 nuclear magnetic resonance spectroscopy

Legerton¹,

Kanamori²,

Weiss³

et al. 1983

Biochemistry

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The nitrogen-15 chemical shift of the N1 (tau)-nitrogen of 15N-labeled histidine and the half-height line widths of proton-coupled resonances of the delta- and omega,omega'-nitrogens of 15N-labeled arginine and of the alpha-nitrogens of 15N-labeled alanine and proline were measured in intact mycelia of Neurospora crassa to obtain to estimates of intracellular pH. For intracellular 15N-labeled histidine, the N1 (tau)-nitrogen chemical shift was 200.2 ppm. In vitro measurements showed that the chemical shift was slightly affected by the presence of phosphate, with which the basic amino acids may be associated in vivo. These considerations indicate a pH of 5.7-6.0 for the environment of intracellular histidine. The half-height line widths of the delta- and omega,omega'-nitrogens of [15N]arginine were 15 and 26 Hz, respectively. In vitro studies showed that these line widths also are influenced by the presence of phosphate, and, after suitable allowance for this, the line widths indicate pH 6.1-6.5 for intracellular arginine. The half-height line widths for intracellular alanine and proline were 17 and 12 Hz, respectively, which are consistent with an intracellular pH of 7.1-7.2. Pools of histidine and arginine are found principally in the vacuole of Neurospora, most likely in association with polyphosphates. Proline and alanine are cytoplasmic. The results reported here are consistent with these localizations and indicate that the vacuolar pH is 6.1 +/- 0.4 while the cytoplasmic pH is 7.15 +/- 0.10. Comparisons of these estimates with those obtained by other techniques and their implications for vacuolar function are discussed.

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“…On the other hand, the 51 residues N-terminal part of the ~rotein shows narrow 1~ resonance lines ( Figure 1) and is quite suitable for conducting ~ NMR studies of specific interaction of the lac repressor with its operator . On the basis of their chemical shift, they were tentatively assigned to 1~ of arginine residues (45)(46)(47)(48). The glutamine and asparagine side-chain 1~2 groups appear as two peaks at a common 15 N chemical shift as indicated by the horizontal lines.…”

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Two-Dimensional¹H,¹⁵N NMR investigation of uniformly¹⁵N-labeledLacRepressor Headpiece

Lancelot

Gervais

Maurizot

1992

Journal of Biomolecular Structure and Dynamics

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15N uniformly labeled lac repressor and lac repressor headpiece were prepared. 15N NMR spectra of lac repressor were shown resolution inadequate for detailed study while the data showed that the 15N labeled N-terminal part of the protein is quite suitable for this type of study allowing future investigation of the specific interaction of the lac repressor headpiece with the lac operator. We report here the total assignment of proton 1H and nitrogen 15NH backbone resonances of this headpiece in the free state. Assignments of the 15N resonances of the protein were obtained in a sequential manner using heteronuclear multiple quantum coherence (HMQC), relayed HMQC nuclear Overhauser and relayed HMQC-HOHAHA spectroscopy. More than 80 per cent of residues were assigned by their 15NH(i)-N1H(i + 1) and 15NH(i)-N1H(i - 1) connectivities. Values of the 3JNH alpha splitting for 39 of the 51 residues of the headpiece were extracted from HMQC and HMQC-J. The observed 15NH(i)-C beta H cross peaks and the 3JNH alpha coupling constants values are in agreement with the three alpha-helices previously described [Zuiderweg, E.R.P., Scheek, R.M., Boelens, R., van Gunsteren, W.F. and Kaptein, R., Biochimie 67, 707 (1985)]. The 3JNH alpha coupling constants can be now used for a more confident determination of the lac repressor headpiece. From these values it is shown that the geometry of the ends of the second and third alpha-helices exhibit deviation from the canonical alpha-helix structure. On the basis of NOEs and 3JNH alpha values, the geometry of the turn of the helix-turn-helix motif is discussed.

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Nitrogen‐15 n.m.r. studies of ¹³C, ¹⁵N labeled arginine

Cited by 24 publications

References 19 publications

Arginine: Its pK_a value revisited

Arginine: Its pK_a value revisited

Measurements of cytoplasmic and vacuolar pH in Neurospora using nitrogen-15 nuclear magnetic resonance spectroscopy

Two-Dimensional¹H,¹⁵N NMR investigation of uniformly¹⁵N-labeledLacRepressor Headpiece

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Nitrogen‐15 n.m.r. studies of 13C, 15N labeled arginine

Cited by 24 publications

References 19 publications

Arginine: Its pKa value revisited

Arginine: Its pKa value revisited

Measurements of cytoplasmic and vacuolar pH in Neurospora using nitrogen-15 nuclear magnetic resonance spectroscopy

Two-Dimensional1H,15N NMR investigation of uniformly15N-labeledLacRepressor Headpiece

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Nitrogen‐15 n.m.r. studies of ¹³C, ¹⁵N labeled arginine

Arginine: Its pK_a value revisited

Arginine: Its pK_a value revisited

Two-Dimensional¹H,¹⁵N NMR investigation of uniformly¹⁵N-labeledLacRepressor Headpiece