Abstract
The amplitude of thermodynamic fluctuations in biological macromolecules determines their conformational behavior, dimensions, nature of phase transitions and effectively their specificity and affinity, thus contributing to fine-tuned molecular recognition. Unique among large-scale conformational changes in proteins are temperature-induced collapse transitions in intrinsically disordered proteins (IDPs). Here, we show that CytR DNA-binding domain, an IDP that folds on binding DNA, undergoes a coil-to-globule transition with temperature in the absence of DNA while exhibiting energetically decoupled local and global structural rearrangements, and maximal thermodynamic fluctuations at the optimal bacterial growth temperature. The collapse is shown to be a continuous transition through a combination of statistical-mechanical modeling and all-atom implicit solvent simulations. Surprisingly, CytR binds single-site cognate DNA with negative cooperativity, described by Hill coefficients less than one, resulting in a graded binding response. We show that heterogeneity arising from varying binding-competent CytR conformations or orientations at the single-molecular level contributes to negative binding cooperativity at the level of bulk measurements due to the conflicting requirements of collapse transition, large fluctuations and folding-upon-binding. Our work reports strong evidence for functionally driven thermodynamic fluctuations in determining the extent of collapse and disorder with implications in protein search efficiency of target DNA sites and regulation.
Highlights
Thermodynamic fluctuations of macromolecules are one of the primary factors driving transcription, binding reactions, allostery and the cellular responses to varied environmental conditions [1,2,3,4,5,6,7]
To quantify the extent to which the overall dimensions of CytR change with temperature, we measured the hydrodynamic radius through non-invasive dynamic light scattering (DLS) experiments that do not require fluorescent probes
The Dynamic light scattering (DLS) experiments were performed at different concentrations of the protein at each temperature from which the effective hydrodynamic radii were extracted
Summary
Thermodynamic fluctuations of macromolecules are one of the primary factors driving transcription, binding reactions, allostery and the cellular responses to varied environmental conditions [1,2,3,4,5,6,7]. Macromolecules are highly sensitive to temperature modulations with the extent of protein oligomerization, degradation and protein disorder all dependent on temperature [8,9] and slaved to solvent composition, structure––extent and strength of hydrogen bonds in the bulk versus the first shell––and motions [10]. It is well established that thermodynamic stability is a continuum ranging from intrinsically disordered (IDPs) to well-folded rigid proteins; experimentally, the measured stability (with reference to the folded state) can be negative (i.e. only unfolded like conformations are populated) to 10–15 RT. In systems that are reasonably well-folded, changes in thermal fluctuations at lower temperatures (
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