fastHDX for IDPs · Revealing the Transient Structures of Intrinsically Disordered Proteins by Microfluidics-Enabled Hydrogen-Deuterium Exchange

Revealing the Transient Structures of Intrinsically Disordered Proteins by Microfluidics-Enabled Hydrogen-Deuterium Exchange

The fastHDX for IDPs project aimed to develop a new technology for the study of the structure and dynamics of proteins that are notoriously challenging to characterise. These proteins are known as intrinsically disordered proteins (IDPs) and contain large regions which have no stable 3D structure. IDPs are heavily implicated in human disease and as such are desirable drug targets. IDPs are not amenable to study by many current analytical techniques as they are too dynamic. Many IDPs only possess transient structure, such as when they bind to their interaction partners, and their structure is therefore difficult to understand and to target with drug molecules. Hydrogen deuterium exchange coupled with mass spectrometry (HDX-MS) is an analytical technique whereby a protein is labelled as a function of time. The incorporation of this label into the protein is dependent on the structural bonds that area of the protein possesses; a more stable, structured region of the protein would uptake the label more slowly than a flexible region of the protein. This means HDX-MS can give insights into protein structure and dynamics. The current commercial, state-of-the-art HDX-MS workflow can probe protein dynamics on a seconds-to-hours timescale and is therefore not applicable to IDPs, whose dynamics occur at a faster rate. This project set out objectives to design, fabricate and validate a microfluidic chip (fastHDX chip) which could be used to label a protein of interest for a very short timepoint, allowing insights into these very fast structural changes. Once the fastHDX chip was tested, the project then followed objectives to use the fastHDX chip to study firstly a well-understood protein, haemoglobin, followed by two proteins of pharmaceutical interest, α-synuclein and CysK as part of the cysteine synthase complex. α-synuclein is heavily implicated in Parkinson’s disease and has no stable structure under its native conditions. The cysteine synthase complex is responsible for the biosynthesis of cysteine in the body and is poorly understood. The project was successful in validating a microfluidic chip which could reproducibly label protein and subsequently quench the labelling reaction on a millisecond-to-second timescale. The fastHDX chip was cheap and easy to fabricate and gave biologically relevant results from testing on haemoglobin. Using the fastHDX chip to study α-synuclein structure revealed the presence of transient structure in one region of the protein. The fastHDX chip was then used to probe the dynamics of the cysteine synthase complex and identified the regions where the subunits bound to each other to form the complex. It also revealed structural changes upon complex formation, enabling a mechanism to be proposed by which a “closed” structure of the protein was formed on complex formation.

Текст от CORDIS, на английски · Данни: CORDIS, © Европейски съюз