High-dimensional NMR methods for intrinsically disordered proteins studies

Grudziąż, Katarzyna; Zawadzka‐Kazimierczuk, Anna; Koźmiński, Wiktor

doi:10.1016/j.ymeth.2018.04.031

Cited by 22 publications

(18 citation statements)

References 63 publications

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“…7; blue) with little dispersion. Such a result is caused by the narrow diversity range of the chemical environments experienced by the observed nuclei [78,79]. The signals of around 6.7 and 7.5 ppm correspond to the side chains of Q and N ( Fig.…”

Section: Nmr Analysismentioning

confidence: 99%

The Intrinsically Disordered Region of GCE Protein Adopts a More Fixed Structure by Interacting With the LBD of the Nuclear Receptor FTZ-F1

Kolonko

Bystranowska

Taube

et al. 2020

Preprint

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The Drosophila melanogaster Germ cell-expressed protein (GCE) is a paralog of the juvenile hormone (JH) receptor - Methoprene tolerant protein (MET). Both proteins mediate JH function, preventing precocious differentiation during D. melanogaster development. Despite that GCE and MET are often referred to as equivalent JH receptors, their functions are not fully redundant and show tissue specificity. Both proteins belong to the family of bHLH-PAS transcription factors. The similarity of their primary structure is limited to defined bHLH and PAS domains, while their long C-terminal fragments (GCEC, METC) show significant differences and are expected to determine differences in GCE and MET protein activities. In this paper we present the structural characterization of GCEC as a coil-like intrinsically disordered protein (IDP) with highly elongated and asymmetric conformation. In comparison to previously characterized METC, GCEC is less compacted, contains more molecular recognition elements (MoREs) and exhibits a higher propensity for induced folding. The NMR shifts perturbation experiment and pull-down assay clearly demonstrated that the GCEC fragment is sufficient to form an interaction interface with the ligand binding domain (LBD) of the nuclear receptor Fushi Tarazu factor-1 (FTZ-F1). Significantly, these interactions can force GCEC to adopt more fixed structure that can modulate the activity, structure and functions of the full-length receptor. The discussed relation of protein functionality with the structural data of inherently disordered GCEC fragment is a novel look at this protein and contributes to a better understanding of the molecular basis of the functions of the C-terminal fragments of the bHLH-PAS family.

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Section: Nmr Analysismentioning

confidence: 99%

The Intrinsically Disordered Region of GCE Protein Adopts a More Fixed Structure by Interacting With the LBD of the Nuclear Receptor FTZ-F1

Kolonko

Bystranowska

Taube

et al. 2020

Preprint

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“…However, the signal dispersion in 2D spectra is often too low to allow effective analysis. Particularly in the case of intrinsically disordered proteins (IDPs), higher‐dimensional techniques are necessary because of extensive signal overlap [6] . Fortunately, there are a number of sparse sampling techniques that make it possible to measure N D spectra faster, [7] even thousands of times faster, depending on the compressibility of the spectrum [8, 9] …”

Section: Introductionmentioning

confidence: 99%

Temperature as an Extra Dimension in Multidimensional Protein NMR Spectroscopy

Shchukina

Małecki

Mateos

et al. 2020

Chemistry A European J

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NMR spectroscopy is a particularly informative method for studying protein structures and dynamics in solution; however, it is also one of the most time‐consuming. Modern approaches to biomolecular NMR spectroscopy are based on lengthy multidimensional experiments, the duration of which grows exponentially with the number of dimensions. The experimental time may even be several days in the case of 3D and 4D spectra. Moreover, the experiment often has to be repeated under several different conditions, for example, to measure the temperature‐dependent effects in a spectrum (temperature coefficients (TCs)). Herein, a new approach that involves joint sampling of indirect evolution times and temperature is proposed. This allows TCs to be measured through 3D spectra in even less time than that needed to acquire a single spectrum by using the conventional approach. Two signal processing methods that are complementary, in terms of sensitivity and resolution, 1) dividing data into overlapping subsets followed by compressed sensing reconstruction, and 2) treating the complete data set with a variant of the Radon transform, are proposed. The temperature‐swept 3D HNCO spectra of two intrinsically disordered proteins, osteopontin and CD44 cytoplasmic tail, show that this new approach makes it possible to determine TCs and their non‐linearities effectively. Non‐linearities, which indicate the presence of a compact state, are particularly interesting. The complete package of data acquisition and processing software for this new approach are provided.

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“…This also applies to intrinsically disordered proteins (IDPs) that display reduced 1 H dispersion due to the lack of permanent secondary or tertiary structure (Figure B) . For large IDPs, the use of high dimensionality or carbon‐detected experiments enhances spectral resolution, notably simplifying the assignment process . Low‐complexity regions (LCRs) in proteins represent a challenge for the above‐mentioned NMR strategies.…”

Section: Introductionmentioning

confidence: 99%

“…[6][7][8] For large IDPs, the use of highd imensionality or carbon-detected experiments en-hances spectralr esolution, notably simplifying the assignment process. [9][10][11] Low-complexity regions (LCRs) in proteins represent ac hallenge for the above-mentioned NMR strategies. LCRs are compositionally biased protein sequences where one or more amino acids are repeated multiple times.…”

Section: Introductionmentioning

confidence: 99%

Site‐Specific Isotopic Labeling (SSIL): Access to High‐Resolution Structural and Dynamic Information in Low‐Complexity Proteins

et al. 2019

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Remarkable technical progress in the area of structural biology has paved the way to study previously inaccessible targets. For example, large protein complexes can now be easily investigated by cryo‐electron microscopy, and modern high‐field NMR magnets have challenged the limits of high‐resolution characterization of proteins in solution. However, the structural and dynamic characteristics of certain proteins with important functions still cannot be probed by conventional methods. These proteins in question contain low‐complexity regions (LCRs), compositionally biased sequences where only a limited number of amino acids is repeated multiple times, which hamper their characterization. This Concept article describes a site‐specific isotopic labeling (SSIL) strategy, which combines nonsense suppression and cell‐free protein synthesis to overcome these limitations. An overview on how poly‐glutamine tracts were made amenable to high‐resolution structural studies is used to illustrate the usefulness of SSIL. Furthermore, we discuss the potential of this methodology to give further insights into the roles of LCRs in human pathologies and liquid–liquid phase separation, as well as the challenges that must be addressed in the future for the popularization of SSIL.

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High-dimensional NMR methods for intrinsically disordered proteins studies

Cited by 22 publications

References 63 publications

The Intrinsically Disordered Region of GCE Protein Adopts a More Fixed Structure by Interacting With the LBD of the Nuclear Receptor FTZ-F1

The Intrinsically Disordered Region of GCE Protein Adopts a More Fixed Structure by Interacting With the LBD of the Nuclear Receptor FTZ-F1

Temperature as an Extra Dimension in Multidimensional Protein NMR Spectroscopy

Site‐Specific Isotopic Labeling (SSIL): Access to High‐Resolution Structural and Dynamic Information in Low‐Complexity Proteins

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