NMR studies of BPTI aggregation by using paramagnetic relaxation reagents

Bernini, Andrea; Spiga, Ottavia; Ciutti, Arianna; Venditti, Vincenzo; Prischi, Filippo; Governatori, M.; Bracci, Luisa; Lelli, Barbara; Pileri, Silvia; Botta, Maurizio; Laschi, Franco; Niccolai, Neri

doi:10.1016/j.bbapap.2006.02.013

Cited by 16 publications

(13 citation statements)

References 23 publications

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“…If access to these surfaces is inhibited on dimerization, the loss of signals in the contact region is reversed. 16 PRE studies on SeR13 were carried out using two different protein concentrations (0.1 and 1.9 mM) in the presence of 1 and 2.5 mM Gd-DTPA respectively. Amide resonances that experienced a protection from Gd- Figure 4(A), where the differences in intensity compared to a standard containing no Gd-DTPA for each condition were normalized and compared to the mean.…”

Section: Concentration-dependent Chemical Shift Studiesmentioning

confidence: 99%

“…The extrapolated RDC values from the two alignment media are then used to determine the symmetry axis of the dimer, allowing the dimer models to be built using the same simple grid search. A paramagnetic perturbation study, [15][16][17][18][19][20] based on shielding of the dimer interface from spin relaxation enhancements has also been added to validate the structures found.…”

Section: Introductionmentioning

confidence: 99%

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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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Section: Concentration-dependent Chemical Shift Studiesmentioning

confidence: 99%

Section: Introductionmentioning

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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“…For identifying a protein-protein binding interface, solvent PREs are measured for the free and complexed forms, where the primary protein of interest is NMR visible (i.e., 15 N and/or 13 C labeled) and the other is NMR invisible (i.e., at natural isotopic abundance). Upon protein-protein complex formation, the previously exposed binding surface will become internally buried, effectively decreasing the solvent-PRE measured for the nuclei at the binding interface (see Figures 1E-G) (Arumugam et al, 1998;Bernini et al, 2006a;Garimella et al, 2006). In theory, the same protocol may be repeated upon reversing the labeling scheme to pinpoint residues from the seconds protein that reside at the interface of the complex.…”

Section: Solvent-paramagnetic Relaxation Enhancement (Pre)mentioning

confidence: 99%

NMR Methods for Structural Characterization of Protein-Protein Complexes

Purslow

Khatiwada

Bayro

et al. 2020

Front. Mol. Biosci.

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Protein-protein interactions and the complexes thus formed are critical elements in a wide variety of cellular events that require an atomic-level description to understand them in detail. Such complexes typically constitute challenging systems to characterize and drive the development of innovative biophysical methods. NMR spectroscopy techniques can be applied to extract atomic resolution information on the binding interfaces, intermolecular affinity, and binding-induced conformational changes in protein-protein complexes formed in solution, in the cell membrane, and in large macromolecular assemblies. Here we discuss experimental techniques for the characterization of protein-protein complexes in both solution NMR and solid-state NMR spectroscopy. The approaches include solvent paramagnetic relaxation enhancement and chemical shift perturbations (CSPs) for the identification of binding interfaces, and the application of intermolecular nuclear Overhauser effect spectroscopy and residual dipolar couplings to obtain structural constraints of protein-protein complexes in solution. Complementary methods in solid-state NMR are described, with emphasis on the versatility provided by heteronuclear dipolar recoupling to extract intermolecular constraints in differentially labeled protein complexes. The methods described are of particular relevance to the analysis of membrane proteins, such as those involved in signal transduction pathways, since they can potentially be characterized by both solution and solid-state NMR techniques, and thus outline key developments in this frontier of structural biology.

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“…(Bernini et al, 2006; Eichmueller and Skrynnikov, 2007; Peterson et al, 2008; Wu et al, 2007; Yang et al, 1996) As a particular example of non-specific short-range PRE perturbation, we cite the work of Lee et al on the weakly associating NESG target, SeR13. (Lee et al, 2010) This study compared Gd-DTPA induced relaxation perturbations at higher and lower protein concentrations to identify the dimer interface.…”

Section: Interaction Interface Determinationmentioning

confidence: 99%

Structural NMR of protein oligomers using hybrid methods

Wang

Lee

Liu

et al. 2011

Journal of Structural Biology

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Solving structures of native oligomeric protein complexes using traditional high resolution NMR techniques remains challenging. However, increased utilization of computational platforms, and integration of information from less traditional NMR techniques with data from other complementary biophysical methods, promises to extend the boundary of NMR-applicable targets. This article reviews several of the techniques capable of providing less traditional and complementary structural information. In particular, the use of orientational constraints coming from residual dipolar couplings and residual chemical shift anisotropy offsets are shown to simplify the construction of models for oligomeric complexes, especially in cases of weak homo-dimers. Combining this orientational information with interaction site information supplied by computation, chemical shift perturbation, paramagnetic surface perturbation, cross-saturation and mass spectrometry allows high resolution models of the complexes to be constructed with relative ease. Non-NMR techniques, such as mass spectrometry, EPR and small angle X-ray scattering, are also expected to play increasingly important roles by offering alternative methods of probing the overall shape of the complex. Computational platforms capable of integrating information from multiple sources in the modeling process are also discussed in the article. And finally a new, detailed example on the determination of a chemokine tetramer structure will be used to illustrate how a non-traditional approach to oligomeric structure determination works in practice.

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NMR studies of BPTI aggregation by using paramagnetic relaxation reagents

Cited by 16 publications

References 23 publications

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

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

NMR Methods for Structural Characterization of Protein-Protein Complexes

Structural NMR of protein oligomers using hybrid methods

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