Engineering Order and Cooperativity in a Disordered Protein.

Structural disorder in proteins arises from a complex interplay between weak hydrophobicity and unfavorable electrostatic interactions. The extent to which the hydrophobic effect contributes to the unique and compact native state of proteins is, however, confounded by large compensation between multiple entropic and energetic terms. Here we show that protein structural order and cooperativity arise as emergent properties upon hydrophobic substitutions in a disordered system with non-intuitive effects on folding and function. Aided by sequence-structure analysis, equilibrium, and kinetic spectroscopic studies, we engineer two hydrophobic mutations in the disordered DNA-binding domain of CytR that act synergistically, but not in isolation, to promote structure, compactness, and stability. The double mutant, with properties of a fully ordered domain, exhibits weak cooperativity with a complex and rugged conformational landscape. The mutant, however, binds cognate DNA with an affinity only marginally higher than that of the wild type, though nontrivial differences are observed in the binding to noncognate DNA. Our work provides direct experimental evidence of the dominant role of non-additive hydrophobic effects in shaping the molecular evolution of order in disordered proteins and vice versa, which could be generalized to even folded proteins with implications for protein design and functional manipulation.

Structural-Energetic Basis for Coupling between Equilibrium Fluctuations and Phosphorylation in a Protein Native Ensemble.

The functioning of proteins is intimately tied to their fluctuations in the native ensemble. The structural-energetic features that determine fluctuation amplitudes and hence the shape of the underlying landscape, which in turn determine the magnitude of the functional output, are often confounded by multiple variables. Here, we employ the FF1 domain from human p190A RhoGAP protein as a model system to uncover the molecular basis for phosphorylation of a buried tyrosine, which is crucial to the transcriptional activity associated with transcription factor TFII-I. Combining spectroscopy, calorimetry, statistical-mechanical modeling, molecular simulations, and in vitro phosphorylation assays, we show that the FF1 domain samples a diverse array of conformations in its native ensemble, some of which are phosphorylation-competent. Upon eliminating unfavorable charge-charge interactions through a single charge-reversal (K53E) or charge-neutralizing (K53Q) mutation, we observe proportionately lower phosphorylation extents due to the altered structural coupling, damped equilibrium fluctuations, and a more compact native ensemble. We thus establish a conformational selection mechanism for phosphorylation in the FF1 domain with K53 acting as a "gatekeeper", modulating the solvent exposure of the buried tyrosine. Our work demonstrates the role of unfavorable charge-charge interactions in governing functional events through the modulation of native ensemble characteristics, a feature that could be prevalent in ordered protein domains.

Slow Folding of a Helical Protein: Large Barriers, Strong Internal Friction, or a Shallow, Bumpy Landscape?

The rate at which a protein molecule folds is determined by opposing energetic and entropic contributions to the free energy that shape the folding landscape. Delineating the extent to which they impact the diffusional barrier-crossing events, including the magnitude of internal friction and barrier height, has largely been a challenging task. In this work, we extract the underlying thermodynamic and dynamic contributions to the folding rate of an unusually slow-folding helical DNA-binding domain, PurR, which shares the characteristics of ultrafast downhill-folding proteins but nonetheless appears to exhibit an apparent two-state equilibrium. We combine equilibrium spectroscopy, temperature-viscosity-dependent kinetics, statistical mechanical modeling, and coarse-grained simulations to show that the conformational behavior of PurR is highly heterogeneous characterized by a large spread in melting temperatures, marginal thermodynamic barriers, and populated partially structured states. PurR appears to be at the threshold of disorder arising from frustrated electrostatics and weak packing that in turn slows down folding due to a shallow, bumpy landscape and not due to large thermodynamic barriers or strong internal friction. Our work highlights how a strong temperature dependence on the pre-exponential could signal a shallow landscape and not necessarily a slow-folding diffusion coefficient, thus determining the folding timescales of even millisecond folding proteins and hints at possible structural origins for the shallow landscape.