Periodic Reporting for period 1 - fastHDX for IDPs (Revealing the Transient Structures of Intrinsically Disordered Proteins by Microfluidics-Enabled Hydrogen-Deuterium Exchange)
Reporting period: 2018-04-01 to 2020-03-31
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.
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.
The fastHDX chip was designed using software packages and contained inlet channels for protein, labelling buffer and quench buffer and an exit channel. The channel lengths, widths and depths were optimised. The fastHDX chip was fabricated entirely from thiol-ene polymer which hardens under UV light. The ratios of the polymer components and the UV curing times were systematically varied to produce a durable fastHDX chip. To mix the liquids on-chip in a highly efficient way, a mixing ‘monolith’ was developed. This was a spatially restricted plug of thiol-ene polymer cured in the microchip channel at the junction between the channels. Two monoliths were placed in each chip to first mix the protein and the labelling buffer together and subsequently the labelled protein and the quench buffer together. The ratio of thiol-ene polymer components, methanol, photoinitiator, curing times and resulting mixing efficiency were adapted and tested to produce fastHDX chips with the ability to mix efficiently.
Once fabricated, the fastHDX chips were validated. The unwanted binding of protein to the fastHDX chip was tested and reduced by adding a coating to the microchip to minimise binding. The mixing of the monoliths was tested using a fluorescent dye and water to track the mixing efficiency. The loss of the HDX label was tested and compared with that of a standard HDX-MS workflow. The repeatability of the fastHDX chips was tested by labelling a protein under identical conditions using 4 different microchips. The robustness of the fastHDX chip was tested by performing the same labelling experiment with a fastHDX chip that was newly fabricated, again once the chip had been used 20 times and again after the chip had been stored for 2 months. The final iteration of the fastHDX chip passed all these validation tests.
The fastHDX chip was then used to study three proteins. First was the well-studied protein haemoglobin. The protein was labelled on-chip at a range of timepoints from 130 milliseconds to 4.3 seconds. The label uptake was then mapped onto the known structure of the protein to give a view of the accuracy of the fastHDX chip labelling. Even at very short labelling times, biologically relevant haemoglobin dynamics could be observed, such as higher uptake in loop regions and lower uptake in stable regions of the protein. The development and validation of the fastHDX chip and the labelling of haemoglobin was disseminated in an open access peer reviewed publication, published on the University of Copenhagen website and presented at 3 international conferences.
The fastHDX chip was then used to study the protein α-synuclein. Labelling on-chip at short timepoints up to 300 milliseconds revealed regions of transient structure in the protein at the C-terminal end. Finally, the fastHDX chip was used to study the protein CysK and its interactions in the cysteine synthase complex. Using a combination of on-chip and manual labelling, binding interactions in the CysK active site were observed, along with significant conformational changes elsewhere in the protein upon complex formation.
Once fabricated, the fastHDX chips were validated. The unwanted binding of protein to the fastHDX chip was tested and reduced by adding a coating to the microchip to minimise binding. The mixing of the monoliths was tested using a fluorescent dye and water to track the mixing efficiency. The loss of the HDX label was tested and compared with that of a standard HDX-MS workflow. The repeatability of the fastHDX chips was tested by labelling a protein under identical conditions using 4 different microchips. The robustness of the fastHDX chip was tested by performing the same labelling experiment with a fastHDX chip that was newly fabricated, again once the chip had been used 20 times and again after the chip had been stored for 2 months. The final iteration of the fastHDX chip passed all these validation tests.
The fastHDX chip was then used to study three proteins. First was the well-studied protein haemoglobin. The protein was labelled on-chip at a range of timepoints from 130 milliseconds to 4.3 seconds. The label uptake was then mapped onto the known structure of the protein to give a view of the accuracy of the fastHDX chip labelling. Even at very short labelling times, biologically relevant haemoglobin dynamics could be observed, such as higher uptake in loop regions and lower uptake in stable regions of the protein. The development and validation of the fastHDX chip and the labelling of haemoglobin was disseminated in an open access peer reviewed publication, published on the University of Copenhagen website and presented at 3 international conferences.
The fastHDX chip was then used to study the protein α-synuclein. Labelling on-chip at short timepoints up to 300 milliseconds revealed regions of transient structure in the protein at the C-terminal end. Finally, the fastHDX chip was used to study the protein CysK and its interactions in the cysteine synthase complex. Using a combination of on-chip and manual labelling, binding interactions in the CysK active site were observed, along with significant conformational changes elsewhere in the protein upon complex formation.
The current state-of-the-art in HDX-MS analysis involves the labelling of proteins at timepoints ranging from 10 seconds to hours. The development of the cheap and easy to fabricate fastHDX chip has allowed the tracking of protein dynamics on a millisecond timescale that was not accessible previously. When used alone, or in conjunction with manual labelling, this can widen the time window that can be probed using HDX-MS. The spatially restricted monoliths allow very efficient on-chip mixing to occur without the need for extra materials or time-consuming milling. To connect the fastHDX chip to commercially available instrumentation, a 3D-printed chip holder was designed. This allows laboratories with standard equipment to use the fastHDX chip with minimal additional costs. The 3D-printed chip holder can withstand the significant pressures associated with the standard HDX-MS workflow.
The impact of this project will be the better understanding of proteins which are challenging to study. Many of these proteins are implicated in diseases such as cancer, Parkinson’s and Alzheimer’s. Expanding our understanding of the way in which these proteins change in structure, for example upon binding to a partner protein, plays an important part in designing drugs which can target these proteins and help to treat their associated disease.
The impact of this project will be the better understanding of proteins which are challenging to study. Many of these proteins are implicated in diseases such as cancer, Parkinson’s and Alzheimer’s. Expanding our understanding of the way in which these proteins change in structure, for example upon binding to a partner protein, plays an important part in designing drugs which can target these proteins and help to treat their associated disease.