13C-detected NMR experiments for automatic resonance assignment of IDPs and multiple-fixing SMFT processing

Abstract

Intrinsically disordered proteins (IDPs) have recently attracted much interest, due to their role in many biological processes, including signaling and regulation mechanisms. High-dimensional 13C direct-detected NMR experiments have proven exceptionally useful in case of IDPs, providing spectra with superior peak dispersion. Here, two such novel experiments recorded with non-uniform sampling are introduced, these are 5D HabCabCO(CA)NCO and 5D HNCO(CA)NCO. Together with the 4D (HACA)CON(CA)NCO, an extension of the previously published 3D experiments (Pantoja-Uceda and Santoro in J Biomol NMR 59:43–50, 2014. doi:10.1007/s10858-014-9827-1), they form a set allowing for complete and reliable resonance assignment of difficult IDPs. The processing is performed with sparse multidimensional Fourier transform based on the concept of restricting (fixing) some of spectral dimensions to a priori known resonance frequencies. In our study, a multiple-fixing method was developed, that allows easy access to spectral data. The experiments were tested on a resolution-demanding alpha-synuclein sample. Due to superior peak dispersion in high-dimensional spectrum and availability of the sequential connectivities between four consecutive residues, the overwhelming majority of resonances could be assigned automatically using the TSAR program. Electronic supplementary materialThe online version of this article (doi:10.1007/s10858-015-9932-9) contains supplementary material, which is available to authorized users.

13C Direct Detected NMR for Challenging Systems

“…These experiments can be performed as 4D experiments (by using a 2D CON as a reference) as well as in the 5D version in which the reference spectrum is the 3D CACON. The proficiency of this CON-CON strategy then led to the design of the analogous experiments based on 1 H direct detection, providing a complete set to meet different experimental needs. − Importantly, this set of spectra is well suited for the implementation of automated protocols for assignment. , …”

A CON-based NMR assignment strategy for pro-rich intrinsically disordered proteins with low signal dispersion: the C-terminal domain of histone H1.0 as a case study

“…33 Thus, for sequential assignment they make use of nuclei with poorer dispersion ( 13 Cα, 13 Cβ or 1 Hα), which may adversely affect the assignment process, particularly in the case of repetitive sequences, such as C-H1.0. To overcome these difficulties some authors proposed the use of 1 H-detected 43,44 or 13 C-detected 23,26,[45][46][47][48] high-dimensionality experiments, 4D or 5D. However, their application to C-H1.0 was problematic because of the sequence peculiarities of C-H1.0 (a sequence with low-amino acid diversity, many repeats, and a relatively high number of Pro residues; see above), together with its very low long-term stability.…”

“…The most successful ones are the 13 C-detected CON-based approaches, which correlate two consecutive CON groups in a protein. [23][24][25][26] Some of them are based on the acquisition of several 5D and/or 4D experiments recorded using non-uniform sampling. 23,25,26 These protocols speed up the assignment process providing backbone and aliphatic chemical shifts, but they require long acquisition times.…”

Non‐Uniform‐Sampling Ultrahigh Resolution TOCSY NMR: Analysis of Complex Mixtures at Microgram Levels

“…The types of sampling schemes include: random sampling, radial sampling and Poisson gap schedule . For processing NUS data, protocols such as maximum entropy (ME) reconstruction, forward maximum entropy (FME) reconstruction, multi‐dimensional decomposition (MDD), sparse multidimensional Fourier transform (SMFT), iterative reweighted least squares (IRLS), low rank (LR), signal separation algorithm (SSA), scrupulous cleaning to remove unwanted baseline artifacts (SCRUB), spectroscopy by integration of frequency‐ and time‐domain information (SIFT), NESTA, and iterative soft threshold (IST) have been reported. In the present manuscript, we have used a combination of Poisson gap schedule with IST processing schemes for the data acquisition and processing, respectively.…”