Quantification of Entropic Excluded Volume Effects Driving Crowding-Induced Collapse and Folding of a Disordered Protein

Abstract

We investigate the conformational properties of the intrinsically disordered DNA-binding domain of CytR in the presence of the polymeric crowder polyethylene glycol (PEG). Integrating circular dichroism, nuclear magnetic resonance, and single-molecule Forster resonance energy transfer measurements, we demonstrate that disordered CytR populates a well-folded minor conformation in its native ensemble, while the unfolded ensemble collapses and folds with an increase in crowder density independent of the crowder size. Employing a statistical−mechanical model, the effective reduction in the accessible conformational space of a residue in the unfolded state is estimated to be 10% at 300 mg/mL PEG8000, relative to dilute conditions. The experimentally consistent PEG−temperature phase diagram thus constructed reveals that entropic effects can stabilize disordered CytR by 10 kJ mol −1 , driving the equilibrium toward folded conformations under physiological conditions. Our work highlights the malleable conformational landscape of CytR, the presence of a folded conformation in the disordered ensemble, and proposes a scaling relation for quantifying excluded volume effects on protein stability.

The Wako-Saitô-Muñoz-Eaton Model for Predicting Protein Folding and Dynamics

“…By introducing the interaction between CytR and its partner DNA, the model successfully describes how, as the partner DNA approaches CytR, the free-energy landscape of CytR in the disordered state with multiple local minima changes into a landscape with a global minimum corresponding to the DNA-bound form of CytR [111,139]. Furthermore, the free-energy landscapes of CytR in the presence of a polymeric crowder, polyethylene glycol (PEG), mimicking crowded intracellular environments, provide a PEG concentration-temperature phase diagram showing that CytR is more folded both at lower temperatures and at higher PEG concentrations, which is in agreement with experimental results [140].…”

Flexible Target Recognition of the Intrinsically Disordered DNA-Binding Domain of CytR Monitored by Single-Molecule Fluorescence Spectroscopy

“…The absolute heat capacity profile of CytR-DBD under low salt conditions displays a broad transition with no excess heat capacity peak . A variable-barrier model analysis of the thermogram demonstrated that the thermal transition occurs as a barrier-less process rather than a barrier-limited transition . The thermally unfolded state of CytR-DBD is highly collapsed and is distinct from the unfolded ensemble at room temperature.…”

“…The increase in unfolded state dimensions at higher urea concentrations is also commonly observed in the unfolded state of many proteins, which could be attributed to the reduction of the hydrophobic effect (as water is a poor solvent). ,− In addition, the changes in chain dimensions are typically associated with the gradual loss of residual secondary structure content. We reported recently that molecular crowding induced by crowding agents like polyethylene glycol (PEG) causes gradual compaction of the unfolded state of CytR-DBD . Taken together, the unfolded state of CytR-DBD is sensitive to changes in solvent conditions, which proves that the balance between various intraprotein and protein–water interactions determines the dimension of the CytR-DBD unfolded state.…”

“…The large-scale structural reordering of CytR-DBD has been investigated by various spectroscopic and theoretical methods. CytR-DBD, while being predominantly disordered, still contains a significant secondary structure, the relative extents of which change gradually depending on solution conditions. ,,, The compact structure reminiscent of the DNA bound form populates as a minor state even in the absence of DNA, , whose relative population increases upon an increase in the solution ionic strength or crowder density. , The heat capacity profile of the disordered CytR-DBD at the low ionic strengths displays a gradual and broad transition; however, at high salt concentrations, an apparent two-state transition with an excess heat capacity peak is observed …”

“…19,39,45,41 The compact structure reminiscent of the DNA bound form populates as a minor state even in the absence of DNA, 40,45 whose relative population increases upon an increase in the solution ionic strength or crowder density. 19,46 The heat capacity profile of the disordered CytR-DBD at the low ionic strengths displays a gradual and broad transition; however, at high salt concentrations, an apparent two-state transition with an excess heat capacity peak is observed. 19 In this work, we have explored the unfolding and the target recognition mechanisms of CytR-DBD using a variant of smFRET spectroscopy, the alternating laser excitation (ALEX) measurements.…”