A primer for carbon‐detected NMR applications to intrinsically disordered proteins in solution

Bastidas, Monique; Gibbs, Eric; Sahu, Debashish; Showalter, Scott A.

doi:10.1002/cmr.a.21327

Cited by 45 publications

(60 citation statements)

References 12 publications

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“…Carbon detect experiments were based on (HA Start) CON-IPAP (16 scans, 1024 × 512 complex data points, with 18 ppm and 36 ppm as 13 C and 15 N sweep widths). 62 …”

Section: Experimental Methodsmentioning

confidence: 99%

Sequence Determinants of the Conformational Properties of an Intrinsically Disordered Protein Prior to and upon Multisite Phosphorylation

et al. 2016

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Many cell signaling events are coordinated by intrinsically disordered protein regions (IDRs) that undergo multisite Serine/Threonine phosphorylation. The conformational properties of these IDRs prior to and following multi-site phosphorylation are directly relevant to understanding their functions. Here, we present results from biophysical studies and molecular simulations that quantify the conformational properties of an 81-residue IDR from the S. cerevisiae transcription factor Ash1. We show that the unphosphorylated Ash1 IDR adopts coil-like conformations that are expanded and well-solvated. This result contradicts inferences regarding global compaction that are derived from heuristics based on amino acid compositions for IDRs with low proline contents. Upon phosphorylation at ten distinct sites, the global conformational properties of pAsh1 are indistinguishable from those of unphosphorylated Ash1. This insensitivity derives from compensatory changes to the pattern of local and long-range intra-chain contacts. We show that the conformational properties of Ash1 and pAsh1 can be explained in terms of the linear sequence patterning of proline and charged residues vis-à-vis all other residues. The sequence features of the Ash1 IDR are shared by many other IDRs that undergo multisite phosphorylation. Accordingly, we propose that our findings might be generalizable to other IDRs involved in cell signaling.

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“…Carbon detect experiments were based on (HA Start) CON-IPAP (16 scans, 1024 × 512 complex data points, with 18 ppm and 36 ppm as 13 C and 15 N sweep widths). 62 …”

Section: Experimental Methodsmentioning

confidence: 99%

Sequence Determinants of the Conformational Properties of an Intrinsically Disordered Protein Prior to and upon Multisite Phosphorylation

et al. 2016

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“…Chemical shift assignments were made using 13 C-Direct Detect methods developed in-house174142, as well as standard 1 H-Detect triple resonance experiments. 13 C and 31 P chemical shifts were referenced to 4,4-dimethyl-4-silapentane-1-sulfonic acid (DSS) and phosphoric acid standards, respectively.…”

Section: Methodsmentioning

confidence: 99%

Phosphorylation induces sequence-specific conformational switches in the RNA polymerase II C-terminal domain

Gibbs

Portz

et al. 2017

Nat Commun

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The carboxy-terminal domain (CTD) of the RNA polymerase II (Pol II) large subunit cycles through phosphorylation states that correlate with progression through the transcription cycle and regulate nascent mRNA processing. Structural analyses of yeast and mammalian CTD are hampered by their repetitive sequences. Here we identify a region of the Drosophila melanogaster CTD that is essential for Pol II function in vivo and capitalize on natural sequence variations within it to facilitate structural analysis. Mass spectrometry and NMR spectroscopy reveal that hyper-Ser5 phosphorylation transforms the local structure of this region via proline isomerization. The sequence context of this switch tunes the activity of the phosphatase Ssu72, leading to the preferential de-phosphorylation of specific heptads. Together, context-dependent conformational switches and biased dephosphorylation suggest a mechanism for the selective recruitment of cis-proline-specific regulatory factors and region-specific modulation of the CTD code that may augment gene regulation in developmentally complex organisms.

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“…In addition, the CaN RD contains three proline residues which are not detected in these spectra. Therefore, we also recorded an (HACA)N(CA)CON NMR experiment, which was instrumental in determining the complete backbone trace for this protein (Bastidas, Gibbs et al 2015). …”

Section: Methods and Experimentsmentioning

confidence: 99%

1H, 15N, and 13C chemical shift assignments of the regulatory domain of human calcineurin

et al. 2017

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Calcineurin (CaN) plays an important role in T-cell activation, cardiac system development and nervous system function. Previous studies have demonstrated that the regulatory domain (RD) of CaN binds calmodulin (CaM) towards the N-terminal end. Calcium-loaded CaM activates the serine/threonine phosphatase activity of CaN by binding to the RD, although the mechanistic details of this interaction remain unclear. It is thought that CaM binding at the RD displaces the auto-inhibitory domain (AID) from the active site of CaN, activating phosphatase activity. In the absence of calcium-loaded CaM, the RD is disordered, and binding of CaM induces folding in the RD. In order to provide mechanistic detail about the Ca…aN interaction, we have undertaken an NMR study of the RD of CaN. Complete 13C, 15N and 1H assignments of the RD of CaN were obtained using solution NMR spectroscopy. The backbone of RD has been assigned using a combination of 13C-detected CON-IPAP experiments as well as traditional HNCO, HNCA, HNCOCA and HNCACB-based 3D NMR spectroscopy. A 15N-resolved TOCSY experiment has been used to assign Hα and Hβ chemical shifts.

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A primer for carbon‐detected NMR applications to intrinsically disordered proteins in solution

Cited by 45 publications

References 12 publications

Sequence Determinants of the Conformational Properties of an Intrinsically Disordered Protein Prior to and upon Multisite Phosphorylation

Sequence Determinants of the Conformational Properties of an Intrinsically Disordered Protein Prior to and upon Multisite Phosphorylation

Phosphorylation induces sequence-specific conformational switches in the RNA polymerase II C-terminal domain

1H, 15N, and 13C chemical shift assignments of the regulatory domain of human calcineurin

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