Effects of Crowding and Environment on the Evolution of Conformational Ensembles of the Multi-Stimuli-Responsive Intrinsically Disordered Protein, Rec1-Resilin: A Small-Angle Scattering Investigation

Abstract

In this study, we explore the overall structural ensembles and transitions of a biomimetic, multi-stimuli-responsive, intrinsically disordered protein (IDP), Rec1-resilin. The structural transition of Rec1-resilin with change in molecular crowding and environment is evaluated using small-angle neutron scattering and small-angle X-ray scattering. The quantitative analyses of the experimental scattering data using a combination of computational models allowed comprehensive description of the structural evolution, organization, and conformational ensembles of Rec1-resilin in response to the changes in concentration, pH, and temperature. Rec1-resilin in uncrowded solutions demonstrates the equilibrium intrinsic structure quality of an IDP with radius of gyration Rg ∼ 5 nm, and a scattering function for the triaxial ellipsoidal model best fit the experimental dataset. On crowding (increase in concentration >10 wt %), Rec1-resilin molecules exert intermolecular repulsive force of interaction, the Rg value reduces with a progressive increase in concentration, and molecular chains transform from a Gaussian coil to a fully swollen coil. It is also revealed that the structural organization of Rec1-resilin dynamically transforms from a rod (pH 2) to coil (pH 4.8) and to globular (pH 12) as a function of pH. The findings further support the temperature-triggered dual-phase-transition behavior of Rec1-resilin, exhibiting rod-shaped structural organization below the upper critical solution temperature (∼4 °C) and a large but compact structure above the lower critical solution temperature (∼75 °C). This work attempted to correlate unusual responsiveness of Rec1-resilin to the evolution of conformational ensembles.

A Sustainable Biomineralization Approach for the Synthesis of Highly Fluorescent Ultra-Small Pt Nanoclusters

“…Also, the maximum molecular dimension (D max ) of the Rec1-resilin in PtNMQCs-Rec1-resilin nanobioconjugates was estimated to be~200.5 Å from the P(r) function, which is slightly less than that of pure Rec1-resilin (~205.5 Å). The obtained structural parameters of pristine Rec1-resilin are in general agreement with previous reports [46,47]. The Rg of pristine Rec1-resilin and nanobioconjugates obtained by P(r) fit was also validated using a shape-independent polymer excluded volume (PEV) model fit ( Figure 3C) [51].…”

“…Rec1-resilin is an IDP which is characterized by conformational heterogeneity and a lack of persistent secondary/tertiary structure, and represents a dynamic structural ensemble [46,47]. Consequently, it continues to be very difficult to investigate the structural heterogeneity and dynamical properties of IDPs, both experimentally and through simulation studies.…”

Features of molecular recognition of intrinsically disordered proteins via coupled folding and binding

“…156,157 Conformational compaction of IDPs/ IDRs by macromolecular crowders has been observed, where the effect depends not only on the crowder size and concentration, but also on the properties of IDPs/IDRs. [158][159][160][161][162][163] MAP2c, p21 Cip1 , and FlgM show global compaction and local structuring in crowded conditions. 164,165 The distal helix of calcineurin and transiently helical regions of ACTR are also stabilized when crowded by synthetic polymers.…”

“…In particular, the malleability of IDPs/IDRs makes them susceptible to the influence of macromolecular crowders . Conformational compaction of IDPs/IDRs by macromolecular crowders has been observed, where the effect depends not only on the crowder size and concentration, but also on the properties of IDPs/IDRs . MAP2c, p21 Cip1 , and FlgM show global compaction and local structuring in crowded conditions .…”

Protein ensembles link genotype to phenotype

“…The conformational space is vast, with the available X-crystal structures covering only a certain fraction, as recently elegantly documented for the Abl tyrosine kinase, for which molecular dynamics simulations and Markov state models identified a protein conformation apparently in a Lilly in-house structure of Abl with WHI-P15 but not in the Protein Data Bank (PDB) [57]. Even though here exploited for drug discovery, it is reasonable to expect that evolution has made use of such vast ensembles as well, adapting them in different ways [58, 59], including in thermostability [60, 61], diverse cellular environments [62], protein disorder and the switches between the ordered and the disordered state [63, 64], detailed linker histone sequence and posttranslational modification (PTM) [65] as well as catalysis of an (O-linked β-N-acetylglucosamine) O-GlcNAc PTM of nuclear and cytosolic protein [66], allosteric interaction networks and signaling pathways [67], and in higher-order organization [68]. This expectation not only can help in prediction of ligand binding [69] but also has inspired the proposition that accounting for conformational heterogeneity and dynamics would benefit protein design methods [70].…”