Solution NMR Structure Determination of Polytopic α-Helical Membrane Proteins

Columbus, Linda; Kroncke, Brett M.

doi:10.1016/bs.mie.2014.12.005

Cited by 3 publications

(1 citation statement)

References 75 publications

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“…Their structures are often difficult to characterize in a membranous environment, which can cloak important interactions with lipids critical to their function. Recent advances in membrane protein structure determination using X-ray crystallography and NMR spectroscopy are significant, [1][2][3][4][5][6][7][8][9][10][11][12][13] in spite of challenges to obtaining a single crystal of target membrane proteins or achieving reasonably fast tumbling of solubilized membrane proteins in solution or well-ordered proteoliposome in the solid state, due to the hydrophobic nature of the transmembrane domains of membrane proteins. Nevertheless, information about long-range distances between helices or domains and depth of insertion remains rare compared to the local structure.…”

mentioning

confidence: 99%

Paramagnetic effects on the NMR spectra of isotropic bicelles with headgroup modified chelator lipids and metal ions

Tang

Mao

et al. 2016

Phys. Chem. Chem. Phys.

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mentioning

confidence: 99%

Paramagnetic effects on the NMR spectra of isotropic bicelles with headgroup modified chelator lipids and metal ions

Tang

Mao

et al. 2016

Phys. Chem. Chem. Phys.

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Towards interpretation of intermolecular paramagnetic relaxation enhancement outside the fast exchange limit

2016

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In an exchanging system between major and minor species, the transverse paramagnetic relaxation enhancement rate observed on the resonances of the major species (Γ 2 (app) ) is dependent upon the exchange regime between the species. Quantitative analysis of PRE data in such systems typically assumes that the overall exchange rate k ex between the species is fast on the PRE time scale (k ex ≫ Γ2). Recently, we have characterized the kinetics of binding of the model protein ubiquitin to large (LUV) and small (SUV) unilamellar lipid-based nanoparticles or liposomes (Ceccon A, Tugarinov V, Bax A, Clore GM (2016). J Am Chem Soc 138:5789-5792). Building upon these results and taking advantage of a strong paramagnetic agent with an isotropic g-tensor, Gd(3+), we were able to measure intermolecular methyl carbon and proton PREs between paramagnetically-tagged liposomes and ubiquitin. In the limit of fast exchange (k ex ≫ Γ2) the ratio of the apparent proton to carbon methyl PREs, ((1)Hm-Γ 2 (app) )/((13)Cm-Γ 2 (app) ), is equal to the square of the ratio of the gyromagnetic ratios of the two nuclei, (γΗ/γC)(2). However, outside the fast exchange regime, under intermediate exchange conditions (e.g. when Γ2 is comparable in magnitude to k ex) the ((1)Hm-Γ 2 (app) )/((13)Cm-Γ 2 (app) ) ratio provides a reliable measure of the 'true' methyl PREs.

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Structural and dynamical basis for the interaction of HSP70-EEVD with JDP Sis1

Matos

Pinheiro

Caruso

et al. 2022

Preprint

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Protein folding, refolding and disaggregation are facilitated by the binding and releasing activity of HSP70 proteins. Such activity is aided by J-domain proteins (JDPs, DNAJs or HSP40s) that stimulate the ATPase activity of HSP70 and stabilize complexes between HSP70 and nonnative proteins. The C-terminus EEVD motif of HSP70 interacts with client proteins and with JDPs. A new understanding of the EEVD interaction with a JDP arises from the results of this work, which gives a detailed NMR characterization of the dynamics and interaction in solution between a class B JDP from yeast, Sis1, and the HSP70-EEVD motif. The EEVD motif binds to multiple sites in Sis1, two of them at the C-terminal domain I (CTDI), the other is a competing interaction between the J-domain and the N-terminal boundary of α-helix 6 (at the GF-region). The interactions at CTDI contributes to the anchoring of HSP70 to Sis1, at site I, and the displacement of the client protein at site II, by competition. These results support a mechanism involved in the regulation of the interaction with HSP70 that is autoinhibitory. Finally, a detailed model of the interaction of the EEVD with the J-domain of Sis1 is proposed.

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Solution NMR Structure Determination of Polytopic α-Helical Membrane Proteins

Cited by 3 publications

References 75 publications

Paramagnetic effects on the NMR spectra of isotropic bicelles with headgroup modified chelator lipids and metal ions

Paramagnetic effects on the NMR spectra of isotropic bicelles with headgroup modified chelator lipids and metal ions

Towards interpretation of intermolecular paramagnetic relaxation enhancement outside the fast exchange limit

Structural and dynamical basis for the interaction of HSP70-EEVD with JDP Sis1

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