A modular approach to map out the conformational landscapes of unbound intrinsically disordered proteins.

Intrinsically disordered proteins (IDPs) fold upon binding to select/recruit multiple partners, morph around the partner's structure, and exhibit allostery. However, we do not know whether these properties emerge passively from disorder, or rather are encoded into the IDP's folding mechanisms. A main reason for this gap is the lack of suitable methods to dissect the energetics of IDP conformational landscapes without partners. Here we introduce such an approach that we term molecular LEGO, and apply it to NCBD, a helical, molten globule­like IDP, as proof of concept. The approach entails the experimental and computational characterization of the protein, its separate secondary structure elements (LEGO building blocks), and their supersecondary combinations. Comparative analysis uncovers specific, yet inconspicuous, energetic biases in the conformational/folding landscape of NCBD, including 1) strong local signals that define the three native helices, 2) stabilization of helix­helix interfaces via soft pairwise tertiary interactions, 3) cooperative stabilization of a heterogeneous three-helix bundle fold, and 4) a dynamic exchange between sets of tertiary interactions (native and nonnative) that recapitulate the different structures NCBD adopts in complex with various partners. Crucially, a tug of war between sets of interactions makes NCBD gradually shift between structural subensembles as a conformational rheostat. Such conformational rheostatic behavior provides a built-in mechanism to modulate binding and switch/recruit partners that is likely at the core of NCBD's function as transcriptional coactivator. Hence, the molecular LEGO approach emerges as a powerful tool to dissect the conformational landscapes of unbound IDPs and rationalize their functional mechanisms.

Downhill (Un)Folding Coupled to Binding as a Mechanism for Engineering Broadband Protein Conformational Transducers.

Canonical proteins fold and function as conformational switches that toggle between their folded (on) and unfolded (off) states, a mechanism that also provides the basis for engineering transducers for biosensor applications. One of the limitations of such transducers, however, is their relatively narrow operational range, limited to ligand concentrations 20-fold below or above their C50. Previously, we discovered that certain fast-folding proteins lose/gain structure gradually (downhill folding), which led us to postulate their operation as conformational rheostats capable of processing inputs/outputs in analog fashion. Conformational rheostats could make transducers with extended sensitivity. Here we investigate this hypothesis by engineering pH transducing into the naturally pH insensitive, downhill folding protein gpW. Particularly, we engineered histidine grafts into its hydrophobic core to induce unfolding via histidine ionization. We designed and tested the effects of ionization via computational modeling and studied experimentally the four most promising single grafts and two double grafts. All tested mutants become reversible pH transducers in the 4-9 range, and their response increases proportionally to how buried the histidine graft is. Importantly, the pH-dependent reversible (un)folding occurs in rheostatic fashion, so the engineered transducers can detect up to 6 orders of magnitude in [H+] for single grafts, and even more for double grafts. Our results demonstrate that downhill (un)folding coupled to binding produces the gradual, analog responses to the ligand (here H+) that are expected of conformational rheostats, and which make them a powerful mechanism for engineering transducers with sensitivity over many orders of magnitude in ligand concentration (broadband).