Impact of Arginine-Phosphate Interactions on the Reentrant Condensation of Disordered Proteins.

Re-entrant condensation results in the formation of a condensed protein regime between two critical ion concentrations. The process is driven by neutralization and inversion of the protein charge by oppositely charged ions. Re-entrant condensation of cationic proteins by the polyvalent anions, pyrophosphate and tripolyphosphate, has previously been observed, but not for citrate, which has similar charge and size compared to the polyphosphates. Therefore, besides electrostatic interactions, other specific interactions between the polyphosphate ions and proteins must contribute. Here, we show that additional attractive interactions between arginine and tripolyphosphate determine the re-entrant condensation and decondensation boundaries of the cationic, intrinsically disordered saliva protein, histatin 5. Furthermore, we show by small-angle X-ray scattering (SAXS) that polyvalent anions cause compaction of histatin 5, as would be expected based solely on electrostatic interactions. Hence, we conclude that arginine-phosphate-specific interactions not only regulate solution properties but also influence the conformational ensemble of histatin 5, which is shown to vary with the number of arginine residues. Together, the results presented here provide further insight into an organizational mechanism that can be used to tune protein interactions in solution of both naturally occurring and synthetic proteins.

Accurate and rapid 3D printing of microfluidic devices using wavelength selection on a DLP printer.

The use of microfluidics on synchrotron X-ray beamlines represents an advanced sample preparation and delivery platform for state-of-the-art X-ray characterization of micro-samples. The recent developments of 3D printing technologies have opened possibilities for rapid fabrication of complex microfluidic devices. One of the major challenges in 3D printing of microfluidic devices using a digital light processing (DLP) desktop printer is that the static liquid resin trapped in the channels, once the "ceiling" is printed, still receives small doses of light through the subsequently printed layers. This easily triggers partial polymerisation of the resin which impedes its flushing out of the channels after completion of the printing session. We show here that it is possible to gain better control over the resin polymerisation and improve the quality of the microfluidic devices by efficiently reducing the penetration depth of the UV LED light through wavelength selection combined with a careful choice of absorber and photo-initiator materials. We produced and tested several structures using a slightly modified desktop printer at 385 nm wavelength with 37 × 37 µm2 pixel resolution at a printed layer thickness of 25 µm. The structures include particle filters, mixers, droplet generators and droplet storage traps with features below 100 µm. We demonstrate crystallisation of model inorganic and organic compounds in trapped droplets and assess the feasibility of in-device X-ray diffraction experiments. This research opens the path for the use of 3D printed microfluidic devices on X-ray beamlines.