Anna Zawadzka-Kazimierczuk

Strategy for complete NMR assignment of disordered proteins with highly repetitive sequences based on resolution-enhanced 5D experiments.

A strategy for complete backbone and side-chain resonance assignment of disordered proteins with highly repetitive sequence is presented. The protocol is based on three resolution-enhanced NMR experiments: 5D HN(CA)CONH provides sequential connectivity, 5D HabCabCONH is utilized to identify amino acid types, and 5D HC(CC-TOCSY)CONH is used to assign the side-chain resonances. The improved resolution was achieved by a combination of high dimensionality and long evolution times, allowed by non-uniform sampling in the indirect dimensions. Random distribution of the data points and Sparse Multidimensional Fourier Transform processing were used. Successful application of the assignment procedure to a particularly difficult protein, δ subunit of RNA polymerase from Bacillus subtilis, is shown to prove the efficiency of the strategy. The studied protein contains a disordered C-terminal region of 81 amino acids with a highly repetitive sequence. While the conventional assignment methods completely failed due to a very small differences in chemical shifts, the presented strategy provided a complete backbone and side-chain assignment.

Non-uniform frequency domain for optimal exploitation of non-uniform sampling.

Random sampling of NMR signal, not limited by Nyquist Theorem, yields up to thousands-fold gain in the experiment time required to obtain desired spectral resolution. Discrete Fourier transform (DFT), that can be used for processing of randomly sampled datasets, provides rarely exploited possibility to introduce irregular frequency domain. Here we demonstrate how this feature opens an avenue to NMR techniques of ultra-high resolution and dimensionality. We present the application of high resolution 5D experiments for protein backbone assignment and measurements of coupling constants from the 4D E.COSY multiplets. Spectral data acquired with the use of proposed techniques allow easy assignment of protein backbone resonances and precise determination of coupling constants.

High-dimensionality 13C direct-detected NMR experiments for the automatic assignment of intrinsically disordered proteins

We present three novel exclusively heteronuclear 5D 13C direct-detected NMR experiments, namely (HN-flipN)CONCACON, (HCA)CONCACON and (H)CACON(CA)CON, designed for easy sequence-specific resonance assignment of intrinsically disordered proteins (IDPs). The experiments proposed have been optimized to overcome the drawbacks which may dramatically complicate the characterization of IDPs by NMR, namely the small dispersion of chemical shifts and the fast exchange of the amide protons with the solvent. A fast and reliable automatic assignment of α-synuclein chemical shifts was obtained with the Tool for SMFT-based Assignment of Resonances (TSAR) program based on the information provided by these experiments.

A set of 4D NMR experiments of enhanced resolution for easy resonance assignment in proteins.

This paper presents examples of techniques based on the principle of random sampling that allows acquisition of NMR spectra featuring extraordinary resolution. This is due to increased dimensionality and maximum evolution time reached. The acquired spectra of CsPin protein and maltose binding protein were analyzed statistically with the aim to evaluate each technique. The results presented include exemplary spectral cross-sections. The spectral data provided by the proposed techniques allow easy assignment of backbone and side-chain resonances.

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

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.

Protonation-dependent conformational variability of intrinsically disordered proteins.

Intrinsically disordered proteins (IDPs) are characterized by substantial conformational plasticity and undergo rearrangements of the time‐averaged conformational ensemble on changes of environmental conditions (e.g., in ionic strength, pH, molecular crowding). In contrast to stably folded proteins, IDPs often form compact conformations at acidic pH. The biological relevance of this process was, for example, demonstrated by nuclear magnetic resonance studies of the aggregation prone (low pH) state of α‐synuclein. In this study, we report a large‐scale analysis of the pH dependence of disordered proteins using the recently developed meta‐structure approach. The meta‐structure analysis of a large set of IDPs revealed a significant tendency of IDPs to form α‐helical secondary structure elements and to preferentially fold into more compact structures under acidic conditions. The predictive validity of this novel approach was demonstrated with applications to the tumor‐suppressor BASP1 and the transcription factor Tcf4.

Generalized Fourier Transform for Non-Uniform Sampled Data

Fourier transform can be effectively used for processing of sparsely sampled multidimensional data sets. It provides the possibility to acquire NMR spectra of ultra-high dimensionality and/or resolution which allow easy resonance assignment and precise determination of spectral parameters, e.g., coupling constants. In this chapter, the development and applications of non-uniform Fourier transform is presented.

High-Dimensional NMR Spectra for Structural Studies of Biomolecules

Recent developments in the acquisition and processing of NMR data sets facilitate the recording of ultra-high-resolution NMR spectra in a reasonable time. The new experiments allow easy resonance assignment for folded and unfolded proteins, as well as the precise determination of spectral parameters, for example, chemical shifts, NOE contacts, coupling constants or cross-correlated relaxation rates. Owing to exceptional resolution of 4D-6D spectroscopy, detailed studies of biomolecules of unprecedented complexity are now possible. Herein, the principles of acquisition and processing methods are presented. The main applications of high-dimensional NMR experiments, including backbone and side-chain resonance assignment in proteins, as well as heteronuclear edited NOE techniques are reviewed.

Amino acid recognition for automatic resonance assignment of intrinsically disordered proteins

Resonance assignment is a prerequisite for almost any NMR-based study of proteins. It can be very challenging in some cases, however, due to the nature of the protein under investigation. This is the case with intrinsically disordered proteins, for example, whose NMR spectra suffer from low chemical shifts dispersion and generally low resolution. For these systems, sequence specific assignment is highly time-consuming, so the prospect of using automatic strategies for their assignment is very attractive. In this article we present a new version of the automatic assignment program TSAR dedicated to intrinsically disordered proteins. In particular, we demonstrate how the automatic procedure can be improved by incorporating methods for amino acid recognition and information on chemical shifts in selected amino acids. The approach was tested in silico on 16 disordered proteins and experimentally on α-synuclein, with remarkably good results.

13 C-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.