High‐Resolution Characterization of Intrinsic Disorder in Proteins: Expanding the Suite of ¹³C‐Detected NMR Spectroscopy Experiments to Determine Key Observables

Abstract

Order in disorder: The characterization of intrinsically disordered proteins by NMR spectroscopy is a necessity on the one hand and a continuous challenge on the other. We propose two experiments that provide diagnostic parameters to monitor the degree of unfolding of a polypeptide. The test was performed on the yeast Cox17 protein, known to gain its function through maturation from an intrinsically disordered state (see figure).

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

“…For proteins like Pdx1-C, which can be studied under mildly acidic conditions, we have found “H N -flip” variants of the 15 N, 13 C-CON experiment to be especially effective, because the increased per-scan sensitivity is coupled to a decrease in the necessary inter-scan delay created by a more rapid recovery of the amide protons to equilibrium. [39, 40] A representative H N -flip 15 N, 13 C-CON pulse sequence is shown in Fig. 2A and forms the basis for constructing the 3D pulse programs we will discuss below.…”

NMR Spectroscopic Studies of the Conformational Ensembles of Intrinsically Disordered Proteins

“…Exploiting improved instrumental sensitivities substantial improvements were made (Chap. 3) due to: (i) non-uniform sampling technologies enabling high-dimensionality (> 4D) experiments (Kazimierczuk et al 2009), (ii) faster acquisition of NMR experiments making use of longitudinal relaxation enhancements (Schanda et al 2006) and (iii) direct heteronuclei ( 13 C) detection using cryoprobe technology (avoiding exchange problems) (Bermel et al 2012;Bertini et al 2011;Novacek et al 2011). The problems of poor signal dispersion and extensive signal overlap found for IDPs are overcome by high-dimensionality spectra (> 4D) using non-uniform sampling (NUS) of indirect dimensions together with appropriate processing schemes, e.g., Sparse Multidimensional Fourier Transform (SMFT) processing (Kazimierczuk et al 2010a;Kazimierczuk et al 2010b;Motackova et al 2010;Zawadzka-Kazimierczuk et al 2012).…”

A suite of amino acid residue type classification pulse sequences for 13C-detected NMR of proteins

“…Initially developed for proteins that contain paramagnetic centers [4][5][6][7][8][9], where direct detection of 13 C helps to reduce relaxation losses, it has many other advantages, that can overcome the inconvenience of the lower sensitivity of the experiments when compared with 1 H-detected NMR spectroscopy. So, 13 C chemical shifts have a much larger dispersion than 1 H chemical shifts, even in the absence of a stable 3D structure, what makes 13 C-detected NMR very useful in the study of completely or partially unfolded proteins [10][11][12][13][14][15][16][17]. The experiments also give information on proline residues, often very abundant in intrinsically disordered proteins.…”