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Downhill Folding Protein Modules as Conformational Rheostats: Roles in Molecular Biology and Applications as Biosensors

Final Report Summary - MOLRHEOSTAT (Downhill Folding Protein Modules as Conformational Rheostats: Roles in Molecular Biology and Applications as Biosensors)

MOLRHEOSTAT aimed at growing the grassroots of quantitative and synthetic molecular biology by investigating novel links between protein folding and functiona via a multidisciplinary, holistic approach that involves biophysical experiments, protein engineering, theory and computation. Traditionally, proteins have been depicted as conformational switches that fold and function by toggling between an on-state (native, active) and an off-state (inactive, undolded) in response to stimuli. However, over the last years we have witnessed the discovery of protein modules that go through constant conformational changes upon unfolding: downhill folding. MOLRHEOSTAT was conceived and implemented with the goal of determining whether downhill folding modules operate mechanistically as conformational rheostats; this is, as single-molecular devices able to continuously modulate signals or responses by gradually fine-tuning their folding conformational ensemble. The concept of conformational rheostats has been applied to the: i) implementation of a general approach for building high-performance, ultrafast, single-molecule sensors based on downhill folding modules; ii) analysis of the roles of conformational rheostats in the regulation of several fundamental processes in molecular biology.
The results on the first area have led to the implementation of a biomolecular engineering toolset to facilitate and standardize the implementation of rheostat-based nanosensors. Such toolbox includes computational methods for identifying new downhill modules, protein engineering protocols for manipulating the folding mechanism and stability of target proteins, and strategies for implementing gradual fluorescence readouts. This toolset has been utilized by the team to develop pH, Calcium and ATP biosensors. The second goal is based on the premise that Nature has exploited the conformational rheostat mechanism on key biological processes that require analogic control at the single molecule level. In this regard, we have investigated three fundamental biological processes under the control of proteins that were conformational rheostat candidates:
i) The peripheral subunit binding domain (PSBD) of the pyruvate dehydrogenase multienzyme complex, with operates as molecular hub for the concerted action of the three enzymes involved in the catalytic cycle. Through experiments and simulations we have demonstrated that PSBD binds to its multiple protein partners by implementing a downhill folding mechanism and marginal intrinsic stability that allows it to bind to multiple partners and potentially synchronize the binding events.
ii) The molecular mechanisms that allow the DNA binding domain of a eukaryotic transcription factor (EnHD) to efficiently track and control its target genes. We found two complementary mechanisms. On the first one EnHD exploits its promiscuous specific binding to colocalize near the genes under its control, which contain long tracts of degenerate versions of its cognate site that we term transcription antennas. The second mechanism enables an efficient search for the target site on genomic DNA by gliding over DNA when is partially disordered (at its physiological conditions).
iii) The mechanical unfolding properties of downhill folding modules that convert them onto molecular springs.
The MOLRHEOSTAT findings confirm the functional and mechanistic roles of conformational rheostats in biology as well as their potential for developing nanobiosensors and other synthetic nanodevices.