Droplet microfluidic hydrogen/deuterium exchange for investigating protein dynamics with millisecond precision exchange

ABSTRACT: 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. Droplet HDX processors were used to measure the fast uptake characteristics of a model peptide. Forward exchange measurements demonstrate D2O labelling to be the rate-limiting step, in essence defining 10 milliseconds as the minimum practical incubation time for proteins in typical physiological conditions. 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. At 10 milliseconds, we could observe significant deuterium uptake within the well-defined EF-hand motifs (Ca2+ binding sites). These findings demonstrate millisecond HDX enabled by droplet microfluidics allows areas of heightened plasticity to be detected within a stably folded protein. Associated dataset: PXD077479

INSTRUMENT(S):

ORGANISM(S): Bos Taurus (bovine)

SUBMITTER: Alistair Bailey  

LAB HEAD: Dr Eamonn

PROVIDER: PXD063880 | Pride | 2025-06-23

REPOSITORIES: Pride