Structural Analysis of Large Protein Complexes Using Solvent Paramagnetic Relaxation Enhancements

Madl, Tobias; Güttler, Thomas; Görlich, Dirk; Sattler, Michael

doi:10.1002/anie.201007168

Cited by 74 publications

(43 citation statements)

References 51 publications

(17 reference statements)

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“…In addition to ligand-induced chemical shift changes, we used our system to characterize the solvent accessibility of residues within the ZM241385-bound A 2A R (with NaCl) using solvent paramagnetic relaxation enhancements (PREs). To do so, we used the soluble paramagnetic Gd 3+ -DTPA probe (Petros et al, 1990), which enhances the relaxation rates of protons in a distance dependent manner within roughly 15 Å of the protein/solvent interface (Madl et al, 2011). By acquiring 1 H/ 13 C TROSY HMQC spectra in the absence and presence of Gd 3+ -DTPA, we could compare peak intensities between the two to establish solvent PRE levels.…”

Section: Resultsmentioning

confidence: 99%

Ligand Modulation of Sidechain Dynamics in a Wild-Type Human GPCR

Clark

Dikiy

Chapman

et al. 2018

Biophysical Journal

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GPCRs regulate all aspects of human physiology, and biophysical studies have deepened our understanding of GPCR conformational regulation by different ligands. Yet there is no experimental evidence for how sidechain dynamics control allosteric transitions between GPCR conformations. To address this deficit, we generated samples of a wild-type GPCR (A 2A R) that are deuterated apart from 1 H/ 13 C NMR probes at isoleucine d1 methyl groups, which facilitated 1 H/ 13 C methyl TROSY NMR measurements with opposing ligands. Our data indicate that low [Na + ] is required to allow large agonist-induced structural changes in A 2A R, and that patterns of sidechain dynamics substantially differ between agonist (NECA) and inverse agonist (ZM241385) bound receptors, with the inverse agonist suppressing fast ps-ns timescale motions at the G protein binding site. Our approach to GPCR NMR creates a framework for exploring how different regions of a receptor respond to different ligands or signaling proteins through modulation of fast ps-ns sidechain dynamics.

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Section: Resultsmentioning

confidence: 99%

Ligand Modulation of Sidechain Dynamics in a Wild-Type Human GPCR

Clark

Dikiy

Chapman

et al. 2018

Biophysical Journal

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“…1). Directly observing the solvent accessible areas is a promising approach not only for mapping interaction surfaces but also for structure determination, as has been shown for proteins 30–32 . Recently, computational protocols using sPRE data were developed for XplorNIH 27 and the Rosetta framework 33 .…”

Section: Introductionmentioning

confidence: 99%

RNA structure refinement using NMR solvent accessibility data

Hartlmüller

Günther

Wolter

et al. 2017

Sci Rep

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NMR spectroscopy is a powerful technique to study ribonucleic acids (RNAs) which are key players in a plethora of cellular processes. Although the NMR toolbox for structural studies of RNAs expanded during the last decades, they often remain challenging. Here, we show that solvent paramagnetic relaxation enhancements (sPRE) induced by the soluble, paramagnetic compound Gd(DTPA-BMA) provide a quantitative measure for RNA solvent accessibility and encode distance-to-surface information that correlates well with RNA structure and improves accuracy and convergence of RNA structure determination. Moreover, we show that sPRE data can be easily obtained for RNAs with any isotope labeling scheme and is advantageous regarding sample preparation, stability and recovery. sPRE data show a large dynamic range and reflect the global fold of the RNA suggesting that they are well suited to identify interaction surfaces, to score structural models and as restraints in RNA structure determination.

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“…PRE-NMR extends the capabilities of traditional NMR approaches to probe protein structure and dynamics by providing long-range distance information up to 30 Å for a proton experiencing relaxation enhancement from a nearby paramagnetic moiety, such as a nitroxide spin label [20]. These long-range constraints can then be used to spatially map intermolecular binding surfaces and position elements within macromolecular complexes [21], [22], [23]. To map the interactions between cTnI-cTnC, nitroxide spin labels were strategically positioned on the intact troponin binary complex (cTnC-cTnI) with the goal of tracking the Ca 2+ sensitive movement of several regions of cTnI (Figure 1C).…”

Section: Introductionmentioning

confidence: 99%

Ca2+-Induced PRE-NMR Changes in the Troponin Complex Reveal the Possessive Nature of the Cardiac Isoform for Its Regulatory Switch

et al. 2014

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The interaction between myosin and actin in cardiac muscle, modulated by the calcium (Ca2+) sensor Troponin complex (Tn), is a complex process which is yet to be fully resolved at the molecular level. Our understanding of how the binding of Ca2+ triggers conformational changes within Tn that are subsequently propagated through the contractile apparatus to initiate muscle activation is hampered by a lack of an atomic structure for the Ca2+-free state of the cardiac isoform. We have used paramagnetic relaxation enhancement (PRE)-NMR to obtain a description of the Ca2+-free state of cardiac Tn by describing the movement of key regions of the troponin I (cTnI) subunit upon the release of Ca2+ from Troponin C (cTnC). Site-directed spin-labeling was used to position paramagnetic spin labels in cTnI and the changes in the interaction between cTnI and cTnC subunits were then mapped by PRE-NMR. The functionally important regions of cTnI targeted in this study included the cTnC-binding N-region (cTnI57), the inhibitory region (cTnI143), and two sites on the regulatory switch region (cTnI151 and cTnI159). Comparison of 1H-15N-TROSY spectra of Ca2+-bound and free states for the spin labeled cTnC-cTnI binary constructs demonstrated the release and modest movement of the cTnI switch region (∼10 Å) away from the hydrophobic N-lobe of troponin C (cTnC) upon the removal of Ca2+. Our data supports a model where the non-bound regulatory switch region of cTnI is highly flexible in the absence of Ca2+ but remains in close vicinity to cTnC. We speculate that the close proximity of TnI to TnC in the cardiac complex is favourable for increasing the frequency of collisions between the N-lobe of cTnC and the regulatory switch region, counterbalancing the reduction in collision probability that results from the incomplete opening of the N-lobe of TnC that is unique to the cardiac isoform.

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Structural Analysis of Large Protein Complexes Using Solvent Paramagnetic Relaxation Enhancements

Cited by 74 publications

References 51 publications

Ligand Modulation of Sidechain Dynamics in a Wild-Type Human GPCR

Ligand Modulation of Sidechain Dynamics in a Wild-Type Human GPCR

RNA structure refinement using NMR solvent accessibility data

Ca2+-Induced PRE-NMR Changes in the Troponin Complex Reveal the Possessive Nature of the Cardiac Isoform for Its Regulatory Switch

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