Time Travel: Flow sculpting for everyone to make movies of proteins in action

Protein motion enables function, with celebrated examples being the oar-like stroke of myosin during muscle contraction, ATP synthase rotation and the valving of ligand-gated ion channels. However, we know almost nothing about how the majority of proteins move and how this contributes to function. This knowledge gap represents an enormous arena for discovery and opportunity to advance biotechnology and develop new drugs.

Examples include the development of new gene editing tools and drugs able to bias protein dynamics toward health.

Protein structural dynamics can be investigated using Hydrogen/Deuterium eXchange (HDX), a technique which is rapidly gaining popularity in UK and global bioscience. HDX involves mixing protein with deuterium for defined periods of seconds to hours, then mixing to preserve incorporated deuterium into the protein backbone, followed by analysis using mass spectrometry (MS) or nuclear magnetic resonance (NMR).

Incorporation rates are used to determine protein structure and dynamics. Currently, however, exchange technology is too slow to observe highly dynamic regions, regions thought to play pivotal roles governing shape-mediated protein function. To observe these behaviours we need methods providing single millisecond or even microsecond incubations, 10,000-fold faster than conventional methods (10 seconds minimum).

To achieve fast HDX, processing in wells is replaced with flow in capillaries or microchannels. Methods using turbulent flow require large protein quantities and cannot achieve single millisecond incubations. Microfluidics in various guises has been explored with each having different failings; uncontrolled incubation times, slow mixing, inability to quench, complex, costly and unreliable fabrication.

To address these shortcomings, we propose a new concept based on advanced flow structuring principles to achieve ultrafast microfluidic HDX. Simulations predict precision time control with microsecond resolution. A device cloning strategy will be used for technology sharing with Jonathan Phillips (Exeter) and Derek Wilson (Toronto), the world-leaders in fast HDX-MS.

An open hardware approach will enable adoption in other labs, expanding the market in readiness for commercialisation.

As proof of concept, the technology will be validated using calmodulin, a protein with well-characterised dynamics and Ca2+-triggered shape-change. The discovery potential will then be test-driven using S100A9, a protein involved in fibril formation leading to neurodegeneration. Millisecond-scale structural transitions are as yet uncharted, but are thought to initiate a fibril competent state.

In summary, we propose to develop, validate, explore and share an ultrafast microfluidic HDX prototype to transform the scale and reach of discovery in the science of protein structural dynamics.