“CON-CON” assignment strategy for highly flexible intrinsically disordered proteins

Abstract

Intrinsically disordered proteins (IDPs) are a class of highly flexible proteins whose characterization by NMR spectroscopy is complicated by severe spectral overlaps. The development of experiments designed to facilitate the sequence-specific assignment procedure is thus very important to improve the tools for the characterization of IDPs and thus to be able to focus on IDPs of increasing size and complexity. Here, we present and describe the implementation of a set of novel ¹H-detected 5D experiments, (HACA)CON(CACO)NCO(CA)HA, BT-(H)NCO(CAN)CONNH and BT-HN(COCAN)CONNH, optimized for the study of highly flexible IDPs that exploit the best resolved correlations, those involving the carbonyl and nitrogen nuclei of neighboring amino acids, to achieve sequence-specific resonance assignment. Together with the analogous recently proposed pulse schemes based on ¹³C detection, they form a complete set of experiments for sequence-specific assignment of highly flexible IDPs. Depending on the particular sample conditions (concentration, lifetime, pH, temperature, etc.), these experiments present certain advantages and disadvantages that will be discussed. Needless to say, that the availability of a variety of complementary experiments will be important for accurate determination of resonance frequencies in complex IDPs.

Linking functions: an additional role for an intrinsically disordered linker domain in the transcriptional coactivator CBP

“…Solution NMR has become the preferred technique for studying IDPs/IDRs 18, 19 as it allows a detailed structural analysis of the polypeptides. To achieve the essentially complete sequence specific assignment (about 95%) of the 407 residues of ID3, including its 75 prolines (18%), the recently developed NMR strategy to study complex IDPs, which combines 1 H and 13 C detected high-resolution multi-dimensional NMR experiments 18, 20–22 was necessary (Supplementary Fig. S1a and Tables S1 and S4).…”

“…By integrating 1 H-detected and 13 C-detected high-resolution multi-dimensional NMR experiments 18, 20–22 we assigned about 95% of backbone chemical shifts, providing atomic resolution information on its structural and dynamic features. An ensemble of possible conformations calculated with experimentally derived ncSP scores and SAXS data supported the presence of several potential binding sites with mild local helical or β propensities, within a largely disordered module.…”

Sparse multidimensional iterative lineshape-enhanced (SMILE) reconstruction of both non-uniformly sampled and conventional NMR data

“…The above methods and a range6 of ingenious related methods have been extensively discussed in a host of recent reviews (Coggins et al 2010; Kazimierczuk et al 2010; Orekhov and Jaravine 2011; Hoch et al 2014; Mobli and Hoch 2014). We note that, from a practical perspective, these methods are rather different from ideas such as projection methods (Eghbalnia et al 2005), GFT (Kim and Szyperski 2003), or the powerful multi-dimensional decomposition method (Orekhov et al 2003), which aim to extract the information most relevant to the spectroscopist from cleverly chosen combinations of time domain data, thereby providing practical access to higher-dimensional (>4) NMR spectroscopy methods (Bermel et al 2013; Piai et al 2014) such as 6D APSY (Fiorito et al 2006). …”

A Set of Efficient nD NMR Protocols for Resonance Assignments of Intrinsically Disordered Proteins

“…Thanks to the introduction of powerful non-uniform sampling and data processing procedures, [36][37][38][39][40] recent studies have demonstrated thatt he increased spectral resolution required in the study of IDPs can be conveniently achieved, without significantly increasing the data acquisition time, by increasingt he number of dimensions in the NMR experiments. [15,18,19,30,32,41,42] Rather than acquiring simple 3D data sets, collection of correlation spectra with higher dimensions would also make it possible to encode the sequential information into the shifts of two chemically different nuclei, for example, ( 15 N i , 1 H i ), andt hereby make the resonance assignments much more robust and reliable, especially in the study of IDPs. AlthoughR Fp ulse schemes providing sequential connectivities via the matching of the 15 N-1 Hc hemical shifts of consecutive residues have been successfully implemented in the literature, it may be sometimes necessary or even advantageous to encodet he sequential information into the shifts of other pair of nuclei such as 15 N-13 CO, 15 N-13 CA and 13 CA-13 CO.…”