Backbone 1H and 15N resonance assignments of the N-terminal SH3 domain of drk in folded and unfolded states using enhanced-sensitivity pulsed field gradient NMR techniques

Zhang, O; Kay, Lewis E.; Olivier, Jean Paul; Forman-Kay, Julie D.

doi:10.1007/bf00398413

Cited by 640 publications

(536 citation statements)

References 28 publications

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“…We attribute this phenomenon to the presence of a preceding Ala residue. Indeed, similar upfield 15N shifts of the backbone amide of Thr have been observed in the only three other reported examples of an Ala-Thr dipeptide sequence within an unfolded protein Logan et al, 1994;Zhang et al, 1994). This effect may arise from a specific contribution of the methyl group of the preceding Ala residue on the I5N shielding of the backbone amide of the following Thr residue (Le & Oldfield, 1994).…”

Section: 'H I3c and I5n Assignments Of Unfolded Gb1supporting

confidence: 74%

Structural and dynamic characterization of the urea denatured state of the immunoglobulin binding domain of streptococcal protein G by multidimensional heteronuclear NMR spectroscopy

Frank¹,

Clore²,

Gronenborn³

1995

Protein Science

132

124

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The structure and dynamics of the urea-denatured B1 immunoglobulin binding domain of streptococcal protein G (GBl) has been investigated by multidimensional heteronuclear NMR spectroscopy. Complete 'H, "N, and I3C assignments are obtained by means of sequential through-bond correlations. The nuclear Overhauser enhancement, chemical shift, and 3JHN, coupling constant data provide no evidence for the existence of any significant population of residual native or nonnative ordered structure. "N relaxation measurements at 500 and 600 MHz, however, provide evidence for conformationally restricted motions in three regions of the polypeptide that correspond to the second P-hairpin, the N-terminus of the a-helix, and the middle of the a-helix in the native protein. The time scale of these motions is longer than the apparent overall correlation time (-3 ns) and could range from about 6 ns in the case of one model to between 4 ps and 2 ms in another; it is not possible to distinguish between these two cases with certainty because the dynamics are highly complex and hence the analysis of the time scale of this slower motion is highly model dependent. It is suggested that these three regions may correspond to nucleation sites for the folding of the GB1 domain. With the exception of the N-and C-termini, where end effects predominate, the amplitude of the subnanosecond motions, on the other hand, are fairly uniform and model independent, with an overall order parameter S2 ranging from 0.4 to 0.5. Keywords: B1 domain; backbone dynamics; I5N relaxation; protein G ; structure; unfolded stateThe two-state model of protein unfolding in chemical denaturants is commonly used to analyze the stability of proteins. In this model, there is an unfolded state and a native state and the relative populations of these states are changed by the addition of the chemical denaturant. The energetics of this transition can be related to structural features of the native state, although, strictly speaking, the energetics depend on the difference in structure between the two states (Dobson, 1992;Shortle, 1993). It is possible that chemically denatured proteins have residual structure, thereby affecting the kinetics and thermodynamics of

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Section: 'H I3c and I5n Assignments Of Unfolded Gb1supporting

confidence: 74%

Structural and dynamic characterization of the urea denatured state of the immunoglobulin binding domain of streptococcal protein G by multidimensional heteronuclear NMR spectroscopy

Frank¹,

Clore²,

Gronenborn³

1995

Protein Science

132

124

View full text Add to dashboard Cite

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“…The distance restraints for SeR13 were derived from 15 N-edited NOESY-HSQC and 13 C-edited NOESY-HSQC spectra using a 1 mM uniformly 13 C, 15 N-enriched SeR13 sample. [25][26][27][28] The locations of the interfacial residues were identified by concentration-dependent chemical shifts and paramagnetic perturbation studies. NOE distances restraints for these residues were carefully assigned to exclude possible intermolecular NOEs.…”

Section: Determining the Monomeric Structure Of Ser13mentioning

confidence: 99%

Three‐dimensional structure of the weakly associated protein homodimer SeR13 using RDCs and paramagnetic surface mapping

et al. 2010

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The traditional NMR-based method for determining oligomeric protein structure usually involves distinguishing and assigning intra-and intersubunit NOEs. This task becomes challenging when determining symmetric homo-dimer structures because NOE cross-peaks from a given pair of protons occur at the same position whether intra-or intersubunit in origin. While there are isotope-filtering strategies for distinguishing intra from intermolecular NOE interactions in these cases, they are laborious and often prove ineffectual in cases of weak dimers, where observation of intermolecular NOEs is rare. Here, we present an efficient procedure for weak dimer structure determination based on residual dipolar couplings (RDCs), chemical shift changes upon dilution, and paramagnetic surface perturbations. This procedure is applied to the Northeast Structural Genomics Consortium protein target, SeR13, a negatively charged Staphylococcus epidermidis dimeric protein (K d 3.4 6 1.4 mM) composed of 86 amino acids. A structure determination for the monomeric form using traditional NMR methods is presented, followed by a dimer structure determination using docking under orientation constraints from RDCs data, and scoring under residue pair potentials and shape-based predictions of RDCs. Validation using paramagnetic surface perturbation and chemical shift perturbation data acquired on sample dilution is also presented. The general utility of the dimer structure determination procedure and the possible relevance of SeR13 dimer formation are discussed.

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“…A selective water flip-back pulse was incorporated to ensure minimum perturbation of the water magnetization (Zhang et al, 1994). The protein sample was 0.7 mM uniformly labeled I5N-CBDce, dissolved in 50 mM sodium acetate, pH 4.0, 10% (vlv) D20.…”

Section: Nmr Spectroscopymentioning

confidence: 99%

Probing the role of tryptophan residues in a cellulose‐binding domain by chemical modification

et al. 1996

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The cellulose-binding domain (CBD,,,) of the mixed function glucanase-xylanase Cex from Cellulomonas fimi contains five tryptophans, two of which are located within the p-barrel structure and three exposed on the surface (Xu GY et al., 1995, Biochemistry 346993-7009). Although all five tryptophans can be oxidized by N-bromosuccinimide (NBS), stopped-flow measurements show that three tryptophans react faster than the other two. NMR analysis during the titration of CBDcex with NBS shows that the tryptophans on the surface of the protein are fully oxidized before there is significant reaction with the two buried tryptophans. Additionally, modification of the exposed tryptophans does not affect the conformation of the backbone of CBDcex, whereas complete oxidation of all five tryptophans denatures the polypeptide. The modification of the equivalent of one and two tryptophans by NBS reduces binding of CBDcex to cellulose by 70% and 90%, respectively. This confirms the direct role of the exposed aromatic residues in the binding of CBDcex to cellulose. Although adsorption to cellulose does afford some protection against NBS, as evidenced by the increased quantity of NBS required to oxidize all of the tryptophan residues, the polypeptide can still be oxidized completely when adsorbed. This suggests that, whereas the binding appears to be irreversible overall [Ong E et al., 1989, Bio/Technology 76044071, each of the exposed tryptophans interacts reversibly with cellulose.

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Backbone 1H and 15N resonance assignments of the N-terminal SH3 domain of drk in folded and unfolded states using enhanced-sensitivity pulsed field gradient NMR techniques

Cited by 640 publications

References 28 publications

Structural and dynamic characterization of the urea denatured state of the immunoglobulin binding domain of streptococcal protein G by multidimensional heteronuclear NMR spectroscopy

Structural and dynamic characterization of the urea denatured state of the immunoglobulin binding domain of streptococcal protein G by multidimensional heteronuclear NMR spectroscopy

Three‐dimensional structure of the weakly associated protein homodimer SeR13 using RDCs and paramagnetic surface mapping

Probing the role of tryptophan residues in a cellulose‐binding domain by chemical modification

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