Direct measurements of transition paths in the folding of single biomolecules using force spectroscopy

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Biological molecules self-assemble (fold') into complex structures. Folding is critical because structure and function are tightly linked, but much remains to be learned about mechanisms and how folding occurs as a physical process. Critical mechanistic information is contained in the transition paths (TPs), the brief moments when the molecule passes through the unstable states that separate the folded and unfolded states, but TPs are hard to detect owing to their brevity. This proposal seizes a timely opportunity offered by a major breakthrough we made last yearthe first direct observation of TPs, studying single molecules under tension using optical tweezersto open a new frontier in folding by exploiting the unique information about microscopic dynamics encoded in TPs. The program focuses on 3 themes:1) Analysis of TP shapes. TP shapes encode much information that has not yet been studied. We will use TP velocities to test physical theories of folding as a diffusive search over an energy landscape, devising a robust measurement of the diffusivity (the key parameter connecting kinetics to thermodynamics). We will characterise transition states directly via pauses in TPs and test microscopic theories of folding that have been impossible to validate. We will distinguish different TP types reflecting different paths through the transition states, validating the multi-pathway view of folding.2) TP properties in different molecules. We will expand the set of molecules studied to establish how TPs reflect molecular properties. DNA hairpins engineered with specific properties (barrier height/position, roughness) will be followed by molecules with more complex folds (RNA pseudoknots, proteins with , , or / structures) to explore how fold topology affects TP properties. We will also probe how evolution shapes folding at the microscopic level by comparing TPs in proteins that have undergone natural selection to TPs in engineered proteins that have not, and by comparing TPs in modern proteins to TPs in ancestral reconstructions at different evolutionary eras.3) Expanded measurements. We will improve our time resolution to enable higher-precision studies of TPs in a wider range of molecules than now possible. We will also extend measurements from the equilibrium regime into the non-equilibrium regime, thereby exploring different parts of the landscape with different properties. Finally, we will examine how TPs change when the axis of the applied force is changed, probing changes in the barriers, exploring how the diffusivity reflects changes in the projection of the energy landscape onto the pulling axis, and quantifying which pulling axes work best as reaction coordinates.This work will reveal previously unexplored facets of folding, test basic concepts from the theory of folding, validate or falsify long-held assumptions, and solidify our understanding of folding as a physical process.