Crowder-directed interactions and conformational dynamics in multistimuli-responsive intrinsically disordered protein

Abstract

The consequences of crowding on the dynamic conformational ensembles of intrinsically disordered proteins (IDPs) remain unresolved because of their ultrafast motion. Here, we report crowder-induced interactions and conformational dynamics of a prototypical multistimuli-responsive IDP, Rec1-resilin. The effects of a range of crowders of varying sizes, forms, topologies, and concentrations were examined using spectroscopic, spectrofluorimetric, and contrast-matching small- and ultrasmall-angle neutron scattering investigation. To achieve sufficient neutron contrast against the crowders, deuterium-labeled Rec1-resilin was biosynthesized successfully. Moreover, the ab initio "shape reconstruction" approach was used to obtain three-dimensional models of the conformational assemblies. The IDP revealed crowder-specific systematic extension and compaction with the level of macromolecular crowding. Last, a robust extension-contraction model has been postulated to capture the fundamental phenomena governing the observed behavior of IDPs. The study provides insights and fresh perspectives for understanding the interactions and structural dynamics of IDPs in crowded states.

Fig. 1.. SANS results of synthesized pure D-Rec1.

(A) SANS intensity profile of D-Rec1 at 3.0 wt % and (B) concentration normalized zero scattering intensity as a function of D₂O%. (C) Corresponding Kratky plot. (D) Model fits to SANS data presented with intensity offset for clarity. (E) Intrinsic disorder profile of Rec1-resilin generated by a database of disordered protein predictions (D²P²) platform. The nine color bars at the top represent disordered regions in Rec1-resilin sequence predicted by different disorder predictors (Espritz, IUPred, etc.). The green bar at the bottom represents predicted disorder region agreement between the nine predictors. The yellow bar at the bottom corresponds to disorder-based binding sites known as molecular recognition features (MoRFs).

Fig. 2.. SANS results of D-Rec1 under macromolecular crowding conditions.

SANS intensity (normalized) profile of D-Rec1 at 3.0 wt % as a function of different crowding agents (contrast matched to solvent) at different concentrations: (A) glucose (GLU), (B) glutathione (GSH), (C) polyethylene glycol (PEG3), (D) Ficoll (FIC70), and (E) dextran (DEX70). (F) Estimated radius of gyration (R_g) of D-Rec1 (using the polymer excluded volume model fit) as a function of crowding agent concentration.

Fig. 3.. Distance distribution results of D-Rec1 under macromolecular crowding conditions.

P(r) curve of D-Rec1 at 3.0 wt % as a function of different crowding agents (contrast matched to solvent) at different concentrations: (A) GLU, (B) GSH, (C) PEG3, (D) FIC70, and (E) DEX70. (F) Estimated R_g of D-Rec1 (using the polymer excluded volume model fit) as a function of crowding agent concentration.

Fig. 4.. Ab initio shape simulations of D-Rec1 under macromolecular crowding conditions.

One among infinite possible ensemble shapes of D-Rec1 as a function of different crowding agents, such as GLU, GSH, PEG3, FIC70, and DEX70 at different concentrations, is presented. Reconstruction was performed using the GASBOR program with the pair-distance distribution output and visualized using the Chimera program. The results of 20 independent simulations were averaged and filtered to give the most probable shape shown as an envelope structure.

Fig. 5.. Crowder-induced aggregation results of D-Rec1.

(A) Optical density (OD), (B) optical images, and (C) combined SANS and USANS intensity profile of pristine D-Rec1 at 3.0 wt %, crowding agents (GLU, GSH, PEG3, FIC70, and DEX70), and their mixtures as a function of crowding agent concentration.

Fig. 6.. Spectrofluorimetric assessment of the effect of crowders on conformational adjustment and surface hydrophobicity change using ANS as the extrinsic fluorescence probe and Tyr as the intrinsic fluorescence probe.

(A) Steady-state fluorescence emission spectra of pure Rec1-resilin and ANS/Rec1-resilin complexes at various molar ratios. (B) Change in λ_max with increase in concentration of ANS in fixed Rec1-resilin. The maximum fluorescence intensity for the complex was observed at approximately ~42.7:1 ANS/Rec1-resilin ratio. (C) Fluorescence spectra of pure ANS and ANS/Rec1 complex with increasing Rec1 concentration. (D) Klotz plot: binding isotherm representing the number of moles of ANS bound per mole of protein. (E) Scatchard treatment of the ANS binding data. (F) Hill plot of the binding data. (G) Enhancement in fluorescence intensity (ΔF) due to ANS bound to Rec1-resilin. (H) Effect of type and level of crowder on the Stern-Volmer quenching constants, K_SV. a.u., arbitrary units.

Fig. 7.. Effect of crowders on the CD spectrum of Rec1-resilin.

(A) CD spectrum of pure Rec1-resilin and pure crowding agents. CD spectra of Rec1-resilin as a function of different crowding agents at different concentrations: (B) GLU, (C) GSH, (D) PEG3, (E) FIC70, and (F) DEX70. High concentrations of crowding agents (10% GSH and 40% others) resulted in high tension (HT) signal greater than 800 V (i.e., the noise becomes disproportionate to the signal) in the middle-UV range (<240 nm) for GSH and in the far-UV range (<200 nm) for GLU, PEG3, FIC70, and DEX70.

Fig. 8.. Crowder-induced conformational dynamics of D-Rec1.

(A) Concentration normalized zero scattering intensity of D-Rec1 as a function of crowder volume fraction and (B) R_g scaled to the pristine D-Rec1 protein [i.e., R_g/R_g(0)] as a function of crowder volume fraction. Self-crowding conformations for Rec1-resilin protein reported elsewhere (23) are given to indicate known packing regions.

Fig. 9.. Energy landscape, schematic of model elements, and potential energy for predicted scenarios.

(A) Schematic of energy landscape showing known and possible routes of conformational change in response to the environment. Dotted lines indicate uniform path of discrete jumps between minimum energy states, and solid lines indicate ensemble behavior. (B) Depiction of the elements of the model used. The resilin molecule is represented as a long chain of nodes linked by rigid segments. A node can represent a single amino acid or a cluster of amino acids. Nodes interact with each other via van der Waals and electrostatic forces. The crowder molecule is represented as a uniform sphere that interacts with the resilin nodes via van der Waals forces. (C) Potential energy between two nodes of a segment for three scenarios: (1) pristine state with no crowders, (2) extended state with few crowders, and (3) contracted state with many crowders.

Fig. 10.. Crowder-induced conformation and energy changes in Rec1-resilin.

(A) R_g of D-Rec1 as a function of crowder volume fraction for different crowders (GLU, GSH, PEG3, FIC70, and DEX70). Solid color lines represent the model predictions and open symbols represent the respective experimental data. Self-crowding conformations for Rec1-resilin protein reported elsewhere (23) are given to further test the model. (B) Model results for an ensemble of states, a snapshot at each crowder volume fraction, for the crowder PEG3. The enthalpy and entropy are calculated for the extreme cases (i.e., contracted and extended states). (C) Corresponding potential energy of the system obtained from the model.