Speeding up sequence specific assignment of IDPs

Abstract

The characterization of intrinsically disordered proteins (IDPs) by NMR spectroscopy is made difficult by the extensive spectral overlaps. To overcome the intrinsic low-resolution of the spectra the introduction of high-dimensionality experiments is essential. We present here a set of high-resolution experiments based on direct (13)C-detection which proved useful in the assignment of α-synuclein, a paradigmatic IDP. In particular, we describe the implementation of 4D HCBCACON, HCCCON, HCBCANCO, 4/5D HNCACON and HNCANCO and 3/4D HCANCACO experiments, specifically tailored for spin system identification and backbone resonances sequential assignment. The use of non-uniform-sampling in the indirect dimension and of the H-flip approach to achieve longitudinal relaxation enhancement rendered the experiments very practical.

A six-dimensional alpha proton detection-based APSY experiment for backbone assignment of intrinsically disordered proteins

“…In addition, 13 C-detection is a very powerful approach to increase signal dispersion and reduce line broadening, which is either caused by solvent exchange or conformational exchange (Felli and Brutscher 2009;Skora et al 2010). 13 C-detected NMR experiments (Bermel et al 2006a(Bermel et al , b, 2012aBertini et al 2004;Pervushin and Eletsky 2003;Shimba et al 2004;Takeuchi et al 2010), in particular when combined with non-uniform sampling techniques (Barna et al 1987;Bermel et al 2012bBermel et al , 2013Chylla and Markley 1995;Coggins and Zhou 2006;Holland et al 2011;Kazimierczuk et al 2006;Kazimierczuk and Orekhov 2011;Korzhneva et al 2001;Novacek et al 2011;Orekhov et al 2001), can therefore enable the resonance assignment and structural characterization of large IDPs (Csizmok et al 2008;Narayanan et al 2010;Novacek et al 2013;Zawadzka-Kazimierczuk et al 2012a).…”

Applications of high dimensionality experiments to biomolecular NMR

“…proposed basing on different spectra (e.g. 2D CON [96], 3D HNCO [93,94] or 3D CACON [96][97][98]) as a starting point in the assignment process. When the resulting signals correlate frequencies of neighboring residues (using so-called "linking" dimensions) one can locate chains of connected residues corresponding to parts of a protein sequence.…”

“…This is especially important in the case of disordered proteins, as their spectra are severely crowded. correlating C α(i) , N (i) , C' (i-1) , for 5D NCOCANCO [100], 5D HBCBCANCO, and 5D HNCANCO [97]. An approach using carbon-detected experiments in IDPs was described recently in [101].…”

Recombinant Intrinsically Disordered Proteins for NMR: Tips and Tricks

“…This will allow the use of advanced techniques such as the recently developed multidimensional NMR experiments (Bermel et al 2012). …”

“…The highly flexible nature of IDPs induces extensive conformational averaging, reducing the nuclear chemical shift dispersion (Bertini et al 2012). Taking flexibility to its extreme, chemical shifts progressively collapse to those of random coil polypeptides, causing extensive resonance overlap.…”