Crowding Effects on the Structure and Dynamics of the Intrinsically Disordered Nuclear Chromatin Protein NUPR1

Abstract

The intracellular environment is crowded with macromolecules, including sugars, proteins and nucleic acids. In the cytoplasm, crowding effects are capable of excluding up to 40% of the volume available to any macromolecule when compared to dilute conditions. NUPR1 is an intrinsically disordered protein (IDP) involved in cell-cycle regulation, stress-cell response, apoptosis processes, DNA binding and repair, chromatin remodeling and transcription. Simulations of molecular crowding predict that IDPs can adopt compact states, as well as more extended conformations under crowding conditions. In this work, we analyzed the conformation and dynamics of NUPR1 in the presence of two synthetic polymers, Ficoll-70 and Dextran-40, which mimic crowding effects in the cells, at two different concentrations (50 and 150 mg/ml). The study was carried out by using a multi-spectroscopic approach, including: site-directed spin labelling electron paramagnetic resonance spectroscopy (SDSL-EPR), nuclear magnetic resonance spectroscopy (NMR), circular dichroism (CD), small angle X-ray scattering (SAXS) and dynamic light scattering (DLS). SDSL-EPR spectra of two spin-labelled mutants indicate that there was binding with the crowders and that the local dynamics of the C and N termini of NUPR1 were partially affected by the crowders. However, the overall disordered nature of NUPR1 did not change substantially in the presence of the crowders, as shown by circular dichroism CD and NMR, and further confirmed by EPR. The changes in the dynamics of the paramagnetic probes appear to be related to preferred local conformations and thus crowding agents partially affect some specific regions, further pinpointing that NUPR1 flexibility has a key physiological role in its activity.

Dance with spins: site-directed spin labeling coupled to electron paramagnetic resonance spectroscopy directly inside cells

“…Nowadays, three main families of spin labels are routinely used for in vitro studies (Fig. 2): nitroxyl radical derivatives (Nitroxides [1][2][3]), metals (Gd(III); Cu(II); Mn(II) chelated by high-affinity cages [4] or protein residues like histideines [5]), triarylmethyl radicals (trityl, OX-SLIM [6]). Reviews on the advantages and limits of these spin labels can be found in the literature.…”

“…The first works on local spin-label dynamics appeared between 1989 and 1996. 1–5 From that time on, this approach has been successfully used to study folding-unfolding-aggregation events, 6–8 intermolecular regulations, 9,10 bio-condensates and coacervates, 11–13 changes in solvent accessibility, 14–16 and interaction between biomolecules. 17,18…”

“…The first works on local spin-label dynamics appeared between 1989 and 1996. [1][2][3][4][5] From that time on, this approach has been successfully used to study folding-unfolding-aggregation events, [6][7][8] intermolecular regulations, 9,10 bio-condensates and coacervates, [11][12][13] changes in solvent accessibility, [14][15][16] and interaction between biomolecules. 17,18 SDSL-EPR to study conformational ensembles Doubly labeled biomolecules or complexes of singly labeled molecules can be used for Dipolar EPR experiments.…”

Challenges in describing the conformation and dynamics of proteins with ambiguous behavior

“…Although X-ray crystallography and cryo-electron microscopy may both be able to trap more than one protein conformer of globular proteins, solution techniques are undoubtedly preferred for uncovering the dynamics of flexible proteins, with NMR being the approach that initially highlighted these features in proteins using different types of measurements (chemical shifts, R1, R2, J-couplings, NOEs, and RDCs). Lately, there have also been efforts dedicated to studying the dynamics of flexible and intrinsically disordered proteins (IDPs) in the cellular context using in-cell NMR and EPR spectroscopy, as a protein’s conformational behavior may differ from what is observed in isolation in the test tube ( Gerez, Prymaczok, and Riek, 2020 ; Bonucci et al, 2021 ). However, due to various experimental challenges, these methods have not become widely used in the community of structural biology.…”

Nanomedicines Meet Disordered Proteins: A Shift from Traditional Materials and Concepts to Innovative Polymers

“…These molecules self-assemble into micelle nanocarriers for the encapsulation of chemotherapeutics or other drugs [ 27 ], and they may exhibit a pH-induced phase transition from a low-dispersity spherical shape to an elongated worm-like one [ 26 ], representing a potential mechanism for the hold-and-release of cargoes. In fact, IDP-based constructs have unique properties of responsiveness towards pH, temperature, and molecular crowding conditions [ 28 ], which are key factors in the microenvironment of diseased tissues. IDP regions can also form nanocages [ 29 ] or be attached to the surface of conjugated nanoparticles [ 30 ], therefore constituting a stealth layer to modulate their circulation half-life.…”