Investigating protein dynamics with millisecond precision using droplet microfluidic hydrogen/deuterium exchange

Hydrogen/deuterium exchange (HDX) methods for studying protein dynamics would benefit from millisecond-scale incubations to probe intrinsically disordered proteins, highly dynamic regions and conformation changes. Here we investigate droplet microfluidics for rapid mixing to trigger D2O labelling, uniform incubations and rapid droplet merging for acid quenching in advance of mass spectrometry. A surfactant-free merging approach combining expansion elements for synchronised droplet collision proved robust. The high diffusive flux of D2O and protons enable microsecond mixing to trigger and arrest D2O labelling, respectively, affording the possibility of single millisecond incubations. Millisecond droplet HDX processors were used to measure forward exchange and demonstrate that D2O labelling is the rate-limiting step, in essence defining 10 milliseconds as the minimum practical incubation time. With the ability to access millisecond time scales the fast dynamics of calmodulin, a model of calcium-triggered allostery with rapid conformational switching, was investigated. Calcium binding increases D2O accessibility to the linker region, reporting the flexibility which enables the scissor-like motion of calmodulin for capturing proteins. The millisecond precision of droplet microfluidic HDX paves the way to advance our understanding of protein structural dynamics.