Generating NMR chemical shift assignments of intrinsically disordered proteins using carbon-detected NMR methods

Abstract

There is an extraordinary need to describe the structures of intrinsically disordered proteins (IDPs) due to their role in various biological processes involved in signaling and transcription. However, general study of IDPs by NMR spectroscopy is limited by the poor 1H-amide chemical shift dispersion typically observed in their spectra. Recently, 13C direct-detected NMR spectroscopy has been recognized as enabling broad structural study of IDPs. Most notably, multi-dimensional experiments based on the 15N,13C-CON spectrum make complete chemical shift assignment feasible. Here we document a collection of NMR based tools that efficiently lead to chemical shift assignment of IDPs, motivated by a case study of the C-terminal disordered region from the human pancreatic transcription factor Pdx1. Our strategy builds on the combination of two 3D experiments, (HN-flip)N(CA)CON and 3D (HN-flip)N(CA)NCO, that enable daisy-chain connections to be built along the IDP backbone, facilitated by acquisition of amino-acid specific 15N,13C-CON detected experiments. Assignments are completed through carbon-detected, TOCSY based side chain chemical shift measurement. Conducting our study required producing valuable modifications to many previously published pulse sequences, motivating us to announce the creation of a database of our pulse programs, which we make freely available through the web.

Molecular Determinants of Tubulin’s C-Terminal Tail Conformational Ensemble

“…We were not able to resolve this ambiguity using carbon detection experiments due to the lack of sufficiently concentrated samples for these relatively insensitive experiments. 36 Neighbor corrected chemical shift predictions 8 indicated that the C-terminal tails have a slight propensity for β -sheet formation (Figure S2). This result is in contrast to computer simulations showing a bias of the C-terminal tails of human tubulin isoforms toward α -helical conformations when simulated in isolation.…”

Expanding the Proteome of an RNA Virus by Phosphorylation of an Intrinsically Disordered Viral Protein

“…All pulsed field gradients used in the experiments were applied for 1 ms with a sine shape. In all pulse sequences, unless otherwise noted by Sahu et al (25), the 90 band-selective 13 C pulses have the Q5 shape (or time reversed, Q5tr), and the band-selective 13 C 180 pulses use the Q3 shape with durations of 384 and 307 s, respectively. The standard 1 H, 15 N HSQC, C_CON, HNCO, and HNCACO experiments were used from the TopSpin pulse program library.…”

“…Briefly, several of the proline resonances were uniquely assigned by their presence in the Ala-specific CON, Ser-specific CON, and TAVI-specific CON spectra. Additional resonance assignments were generated from (H Nflip)N(CA)CON and (H N -flip)N(CA)NCO spectra (25). All NMR data were processed in TopSpin version 2.1 and converted to Sparky (26) format for data analysis.…”

“…We therefore built our NMR strategy around the 15 N, 13 C CON experiment, using recently developed multidimensional NMR techniques for the analysis and chemical shift assignments of IDPs that do contain resonances corresponding to proline residues (25). We expressed and purified isotopically 15 N-, 13 C-labeled NS5A IDD and acquired double and triple resonance spectra on both the unphosphorylated and PKAphosphorylated proteins.…”

Phosphorylation Induces Sequence-Specific Conformational Switches in the RNA Polymerase II C-Terminal Domain

“…Typically, 80 mM imidazole pH 6.5, 50 mM KCl, 10% glycerol, 2 mM DTT and 10% D 2 NMR Spectra were collected at the Lloyd Jackman NMR facility at the Pennsylvania State University on Bruker Avance-III spectrometers operating at proton frequencies of 500, 600 or 850 MHz equipped with TCI single-axis gradient cryoprobes ( 1 H/ 13 C/ 15 N/ 2 H) with enhanced sensitivity for 1 H and 13 C. Phosphorous-detect experiments were performed on a 500 MHz Bruker Avance-III-HD spectrometer equipped with a broadband (BBO) Prodigy CryoProbe. Chemical shift assignments were made using 13 C-Direct Detect methods developed in-house 17,41,42 , 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.…”