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Lune, F. van; Manning, Lane; Dijkstra, Klaas; Berendsen, Herman J. C.; Scheek, Ruud M.

doi:10.1023/a:1019877830921

Cited by 16 publications

(7 citation statements)

References 13 publications

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“…Such ensembles have previously been shown to reproduce the trends of backbone RDCs along the polypeptide sequence. ,, Because the Flexible -Meccano algorithm represents side chains only by a pseudoatom at the C β position, full coordinates for the C β and H β atoms for all staggered rotamers were generated from the N, C α , and C′ positions using idealized tetrahedral geometry. The alignment tensor for each member k of the ensemble was then calculated on the basis of the assumption of steric exclusion , using an efficient in-house written algorithm . In brief, this algorithm calculates the maximal extension of the molecule for each direction of the unit sphere.…”

Section: Resultsmentioning

confidence: 99%

Side-Chain χ₁ Conformations in Urea-Denatured Ubiquitin and Protein G from ³J Coupling Constants and Residual Dipolar Couplings

et al. 2010

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Current NMR information on side-chain conformations of unfolded protein states is sparse due to the poor dispersion particularly of side-chain proton resonances. We present here optimized schemes for the detection of (3)J(HalphaHbeta), (3)J(NHbeta), and (3)J(C'Hbeta) scalar and (1)D(CbetaHbeta) residual dipolar couplings (RDCs) in unfolded proteins. For urea-denatured ubiquitin and protein G, up to six (3)J-couplings to (1)H(beta) are detected, which define the chi(1) angle at very high precision. Interpretation of the (3)J couplings by a model of mixed staggered chi(1) rotamers yields excellent agreement and also provides stereoassignments for (1)H(beta) methylene protons. For all observed amino acids with the exception of leucine, the chemical shift of (1)H(beta3) protons was found downfield from (1)H(beta2). For most residues, the precision of individual chi(1) rotamer populations is better than 2%. The experimental chi(1) rotamer populations are in the vicinity of averages obtained from coil regions in folded protein structures. However, individual variations from these averages of up to 40% are highly significant and indicate sequence- and residue-specific interactions. Particularly strong deviations from the coil average are found for serine and threonine residues, an effect that may be explained by a weakening of side-chain to backbone hydrogen bonds in the urea-denatured state. The measured (1)D(CbetaHbeta) RDCs correlate well with predicted RDCs that were calculated from a sterically aligned coil model ensemble and the (3)J-derived chi(1) rotamer populations. This agreement supports the coil model as a good first approximation of the unfolded state. Deviations between measured and predicted values at certain sequence locations indicate that the description of the local backbone conformations can be improved by incorporation of the RDC information. The ease of detection of a large number of highly precise side-chain RDCs opens the possibility for a more rigorous characterization of both side-chain and backbone conformations in unfolded proteins.

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Section: Resultsmentioning

confidence: 99%

Side-Chain χ₁ Conformations in Urea-Denatured Ubiquitin and Protein G from ³J Coupling Constants and Residual Dipolar Couplings

et al. 2010

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“…Theoretical RDCs were calculated on the basis of the assumption of steric exclusion , using an efficient in-house written algorithm . In short, the maximal extension of a molecule for each direction of a unit sphere is calculated.…”

Section: Methodsmentioning

confidence: 99%

Sequence-Specific Mapping of the Interaction between Urea and Unfolded Ubiquitin from Ensemble Analysis of NMR and Small Angle Scattering Data

et al. 2012

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The molecular details of how urea interacts with, and eventually denatures proteins, remain largely unknown. In this study we have used extensive experimental NMR data, in combination with statistical coil ensemble modeling and small-angle scattering, to analyze the conformational behavior of the protein ubiquitin in the presence of urea. In order to develop an atomic resolution understanding of the denatured state, conformational ensembles of full-atom descriptions of unfolded proteins, including side chain conformations derived from rotamer libraries, are combined with random sampling of explicit urea molecules in interaction with the protein. Using this description of the conformational equilibrium, we demonstrate that the direct-binding model of urea to the protein backbone is compatible with available experimental data. We find that, in the presence of 8 M urea, between 30 and 40% of the backbone peptide groups bind a urea molecule, independently reproducing results from a model-free analysis of small-angle neutron and X-ray scattering data. Crucially, this analysis also provides sequence specific details of the interaction between urea and the protein backbone. The pattern of urea-binding along the amino-acid sequence reveals a higher level of binding in the central part of the protein, a trend which resembles independent results derived from chemical shift mapping of the urea-protein interaction. Together these results substantiate the direct-binding model and provide a framework for studying the physical basis of interactions between proteins and solvent molecules.

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“…For steric alignment P (Ω) can be calculated from excluded volume effects , (see Figure ) as P ( Ω ) = L − l ( Ω ) 4 π L − ∫ l ( Ω ) d Ω ≈ L − l ( Ω ) 4 π L where L is the distance between two infinite parallel planes and l is the maximal length of the molecule in the direction perpendicular to the planes. Thus the parameter L together with the anisotropy of the molecular shape determines the absolute size of the molecular orientation.…”

Section: Theory and Algorithmsmentioning

confidence: 99%

Ensemble Calculations of Unstructured Proteins Constrained by RDC and PRE Data: A Case Study of Urea-Denatured Ubiquitin

2009

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The detailed, quantitative characterization of unfolded proteins is a largely unresolved task due to the enormous experimental and theoretical difficulties in describing the highly dimensional space of their conformational ensembles. Recently, residual dipolar coupling (RDC) and paramagnetic relaxation enhancement (PRE) data have provided large numbers of experimental parameters on unfolded states. To obtain a minimal model of the unfolded state according to such data we have developed new modules for the use of steric alignment RDCs and PREs as constraints in ensemble structure calculations by the program XPLOR-NIH. As an example, ensemble calculations were carried out on urea-denatured ubiquitin using a total of 419 previously obtained RDCs and 253 newly determined PREs from eight cysteine mutants coupled to MTSL. The results show that only a small number of about 10 conformers is necessary to fully reproduce the experimental RDCs, PREs and average radius of gyration. C(alpha) contacts determined on a large set (400) of 10-conformer ensembles show significant (10-20%) populations of conformations that are similar to ubiquitin's A-state, i.e. corresponding to an intact native first beta-hairpin and alpha-helix as well as non-native alpha-helical conformations in the C-terminal half. Thus, methanol/acid (A-state) and urea denaturation lead to similar low energy states of the protein ensemble, presumably due to the weakening of the hydrophobic core. Similar contacts are obtained in calculations using solely RDCs or PREs. The sampling statistics of the C(alpha) contacts in the ensembles follow a simple binomial distribution. It follows that the present RDC, PRE, and computational methods allow the statistically significant detection of subconformations in the unfolded ensemble at population levels of a few percent.

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Cited by 16 publications

References 13 publications

Side-Chain χ₁ Conformations in Urea-Denatured Ubiquitin and Protein G from ³J Coupling Constants and Residual Dipolar Couplings

Side-Chain χ₁ Conformations in Urea-Denatured Ubiquitin and Protein G from ³J Coupling Constants and Residual Dipolar Couplings

Sequence-Specific Mapping of the Interaction between Urea and Unfolded Ubiquitin from Ensemble Analysis of NMR and Small Angle Scattering Data

Ensemble Calculations of Unstructured Proteins Constrained by RDC and PRE Data: A Case Study of Urea-Denatured Ubiquitin

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Cited by 16 publications

References 13 publications

Side-Chain χ1 Conformations in Urea-Denatured Ubiquitin and Protein G from 3J Coupling Constants and Residual Dipolar Couplings

Side-Chain χ1 Conformations in Urea-Denatured Ubiquitin and Protein G from 3J Coupling Constants and Residual Dipolar Couplings

Sequence-Specific Mapping of the Interaction between Urea and Unfolded Ubiquitin from Ensemble Analysis of NMR and Small Angle Scattering Data

Ensemble Calculations of Unstructured Proteins Constrained by RDC and PRE Data: A Case Study of Urea-Denatured Ubiquitin

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Side-Chain χ₁ Conformations in Urea-Denatured Ubiquitin and Protein G from ³J Coupling Constants and Residual Dipolar Couplings

Side-Chain χ₁ Conformations in Urea-Denatured Ubiquitin and Protein G from ³J Coupling Constants and Residual Dipolar Couplings