Hydrogen Exchange of Individual Amide Protons in the E. Coli<i>lac</i>Repressor DNA-binding Domain: A Nuclear Magnetic Resonance Study (29)

Boelens, Rolf; Gros, Piet; Scheek, Ruud M.; Verpoorte, Jacob A.; Kaptein, Robert

doi:10.1080/07391102.1985.10508416

Cited by 15 publications

(17 citation statements)

References 24 publications

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“…The coupling constants are related to the <I> torsion angle of the peptide backbone by the Karplus relationship (43). The Figure 5 shows the correlation between the corrected values of 3 JNHa coupling constants and the secondary structure of the headpiece as it was determined on the basis ofthe NOE (21) and exchange data (44). If one takes into consideration the fact that a-helix structure generates 3 JNHa coupling constant smaller than 5Hz, the agreement with the previous location of the secondary structure appears globally satisfactory.…”

Section: N Resonancesmentioning

confidence: 93%

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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Section: N Resonancesmentioning

confidence: 93%

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

Lancelot

Gervais

Maurizot

1992

Journal of Biomolecular Structure and Dynamics

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“…As suggested by Wagner (1980), the correlation of exchange of two labile protons can be assessed by comparing their exchange rates with decay rates for the mutual NOE between the protons [see also Boelens et al (1985) and Roder et al (1985a,b)]. While the resonance peak intensity monitors directly the 'H content of that site, the NOE difference peak is proportional to the fraction of molecules with both sites 'H labeled.…”

Section: H E and H Exchangementioning

confidence: 98%

Hydrogen exchange of primary amide protons in basic pancreatic trypsin inhibitor: evidence for amino group rotation in buried asparagine side chains

Tüchsen¹,

Woodward²

1987

Biochemistry

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Hydrogen-deuterium exchange is measured for the buried primary amide groups of Asn-43 and Asn-44 in bovine pancreatic trypsin inhibitor. Amide protons trans and cis to the amide carbonyl oxygen (HE and HZ, respectively) exchange at indistinguishable rates. Uncorrelated exchange of HE and HZ is established for both residues by following the nuclear Overhauser enhancement from HE to HZ during the deuterium exchange. The exchange of Asn-43 and Asn-44 side-chain protons differs qualitatively from exchange of primary amide groups in fully solvated model compounds, for which HE generally exchanges faster than HZ. The equal rates for the buried primary amide HE and HZ in BPTI are not a consequence of coupled exchange. The data indicate rapid rotation around the CO-NH2 bond for both Asn-43 and Asn-44 and suggest considerable lability of intramolecular hydrogen bonds. The side chain of Asn-43 has all of its polar atoms integrated into the very stable hydrogen-bonded structure of the protein. Asn-44 is hydrogen-bonded to side chains and to a buried water molecule. Solvent isotope exchange is several orders of magnitude more restricted by protein secondary and tertiary structure than the CO-NH2 rotation, indicating that N delta H2 groups flip many times before hydrogen isotope exchange occurs.

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“…The following considerations prompted us to use NOESY rather than COSY [24] for the studies of NH exchange rates [25]. In contrast to COSY, which produces antiphase crosspeak signals with zero integrated intensity, the NOESY crosspeak heights are defined by the signal amplitudes measured at tl = 0.…”

Section: Methodsmentioning

confidence: 99%

Secondary structure determination for α‐neurotoxin from Dendroaspis polylepis polylepis based on sequence‐specific ¹H‐nuclear‐magnetic‐resonance assignments

Labhardt¹,

Hunziker‐Kwik²,

Wüthrich³

1988

European Journal of Biochemistry

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Sequence-specific assignments are presented for the polypeptide backbone protons and a majority of the amino-acid-side-chain protons of a-neurotoxin from Dendroaspis polylepis polylepis, and individual amide protonexchange rates with the solvent are reported. The secondary structure and the hydrogen-bonding patterns in the regular secondary structure elements are deduced from nuclear Overhauser effects and the sequence locations of the slowly exchanging amide protons. The molecule includes a three-stranded antiparallel P-sheet, and there are indications that two additional short chain segments are arranged in an antiparallel P-sheet. These structural elements are similar, but not identical, to either the secondary structure reported for erabutoxin b in single crystals, or the solution structure of cytotoxin CTXIIb from Naja mossambica mossambica.Neurotoxin from the venom of snakes of the Elapidae family are low-molecular-mass proteins which bind specifically to the nicotinic acetylcholine receptor in cholinergic synapses, thus blocking postsynaptic membrane depolarization. Over 60 homologous snake toxins have been sequenced which can be classified either as short neurotoxins with four disulfide bonds, or long neurotoxins with five disulfide bonds, where the pairing for four disulfides is similar in all the neurotoxins [I -31. Studies of neurotoxin conformation have been pursued with both X-ray diffraction in single crystals and NMR in solution [4 -111. The molecular models, thus obtained, show that the homology of the primary structures carries over to the three-dimensional structures, but there is also evidence for structural disorder in the neurotoxin crystals and for differences between corresponding structures in crystals and in solution. Examples for this include the short neurotoxin erabutoxin b, where there are differences in side-chain orientations and the backbone arrangements of the peripheral loops between two different crystallographic maps [4, 61, and difficulties in the interpretation of the electron density maps near the tip of a long central loop [S]. Furthermore, residue H7 was reported to be on the molecular surface in both crystal structures [4 -71, whereas such an orientation seems to be incompatible with chemical modification studies and NMR measurements in solution [8, 91, which both suggest that H7 is buried in the interior of the protein. For the long neurotoxin a-bungarotoxin there is evidence that the regular P-sheet structures extend over a longer stretch of the polypeptide chain in the structure in solution, determined by NMR, than in the crystal structure [I 11. To Ahhreviations. 1 D,2D, one-dimensional, two-dimensional; COSY, 2D-correlated spectroscopy; 2QF-COSY, two-quantumfiltered COSY; NOESY, 2D nuclear Overhauser enhancement spectroscopy; RELAYED-COSY, 2D-relayed coherence transfer spectroscopy; FID, free-induction decay.tendency of neurotoxins to adopt multiple, different conformations in different environments, we have started an NMR investigation of the short a-neurotoxin f...

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Hydrogen Exchange of Individual Amide Protons in the E. ColilacRepressor DNA-binding Domain: A Nuclear Magnetic Resonance Study (29)

Cited by 15 publications

References 24 publications

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

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

Hydrogen exchange of primary amide protons in basic pancreatic trypsin inhibitor: evidence for amino group rotation in buried asparagine side chains

Secondary structure determination for α‐neurotoxin from Dendroaspis polylepis polylepis based on sequence‐specific ¹H‐nuclear‐magnetic‐resonance assignments

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Hydrogen Exchange of Individual Amide Protons in the E. ColilacRepressor DNA-binding Domain: A Nuclear Magnetic Resonance Study (29)

Cited by 15 publications

References 24 publications

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

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

Hydrogen exchange of primary amide protons in basic pancreatic trypsin inhibitor: evidence for amino group rotation in buried asparagine side chains

Secondary structure determination for α‐neurotoxin from Dendroaspis polylepis polylepis based on sequence‐specific 1H‐nuclear‐magnetic‐resonance assignments

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

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

Secondary structure determination for α‐neurotoxin from Dendroaspis polylepis polylepis based on sequence‐specific ¹H‐nuclear‐magnetic‐resonance assignments