Sofia Jaho's update on her PhD

Can you remind us briefly what your project is about?

My project focuses on the crystallization of membrane proteins with an automated microfluidic pipeline. But what do all these fancy words really mean? They simply describe that my work is associated with the process of crystallizing membrane proteins on microfluidic devices and at the same time I am trying to develop instrumentation to make the whole process more automated and a crystallographer’s life much easier!

What are membrane proteins and why we need to study them? As I described in a previous blogpost, membrane proteins are proteins located on the membranes of the cell. Depending on their structure, they are involved in a plethora of biological functions. For example, there is a family of membrane proteins (transporters) that are responsible for transporting molecules across the cellular membrane. However, in order to understand the function of the various families of membrane proteins, we need to know their 3D structure at high resolution, which means we need to know details of how these proteins look like close to the atomic length scale. Nowadays, we can study the structure of soluble and membrane proteins by using various methods. The most well-established method to do so is X-ray diffraction. But, the premise for that is to have crystals of the targeted membrane protein. And this is the reason why the crystallization of these biological macromolecules is crucial. We need to find the right, optimal conditions for crystallizing each target and use X-ray crystallography to “read” their structure.

However, one might think: how can we optimize the crystallization of membrane proteins and which tools we need? Well, from my point of view the answer is microfluidics! I am working on developing microfluidic chips for crystallizing membrane proteins on them (on-chip crystallization) by using the microdialysis method. When the protein crystals are grown, scientists must harvest them and protect them (usually by cryocooling the crystals) in order to use them for X-ray diffraction measurements (X-rays can be harsh on the fragile protein crystals and lead to what is called radiation damage). When radiation damage occurs, there is not much information that someone can get for the protein’s structure. The microchips that I am developing use materials that are compatible for in situ X-ray measurements. This means that we don’t have to harvest the protein crystals and the crystals won’t be damaged during the steps of harvesting or cryocooling. So basically, we can use one single device in order to crystallize membrane proteins and directly use them for on-chip and in situ X-ray diffraction experiments. Another advantage of microfluidic crystallization is to significantly reduce the amounts of protein samples to a few hundred nanoliters.

Moreover, apart from purifying membrane proteins and trying to crystallize them on my microfluidic devices, I am also working on instrumental development for the automation of the whole process. I am developing a whole microfluidic pipeline (consider it a bench-top platform) where the process of crystallizing membrane proteins will be automated and fully controlled. For example, I have so far integrated a fluidic system for mixing and circulating the crystallization agents into the microchip. At the same time, I have developed a prototype for temperature control that provides thermal regulation of the on-chip crystallization experiments. By establishing temperature and chemical composition control of the on-chip crystallization we will be able to study phase diagrams and develop protein crystals in precise conditions.

What important milestones have you reached until now?

I have been working on microfabrication techniques in order to develop my microchips. I tried various protocols and materials and I started my first tests with the chips. And they worked! First, I performed experiments with soluble proteins. So, I got beautiful Lysozyme and Thaumatin crystals grown on-chip with the dialysis method and I got a close to 2 Å resolution density map of the Lysozyme crystals by performing in situ X-ray diffraction experiments at BM30A-FIP (ESRF).

The next step was to crystallize model membrane proteins on-chip (like AcrB from Escherichia coli, one of the membrane proteins that I am working with at Grenoble) and more challenging protein targets. Towards this end, my RAMP colleagues contributed a lot by providing me samples of their membrane proteins. So, I have crystallized also on-chip with the dialysis method, TmPPase and SERCA provided by Jannik Strauss and Samuel Hjorth-Jensen, respectively. All the above-mentioned membrane proteins were used for in situ X-ray diffraction experiments at PETRA III and ALBA synchrotron sources. Currently, I am working on the optimization of the crystals obtained by these membrane proteins in order to get better diffraction resolution. I should also mention that while I am writing these lines, I am in Gothenburg performing my secondment at AstraZeneca. Our project with AstraZeneca is about using my microchips for crystallizing some of the protein targets that the company is interested in.

At the same time, I was working on the development of the microfluidic platform. I have managed to make it run for visual observation and monitoring of the crystallization process and we have integrated a fluidic system for the automated mixing and circulation of the crystallization solutions. I have also designed and developed a system for controlling the temperature of the chips during the crystallization experiments, but more tests and configurations need to be done before I can claim that the system is fully operating.

We recently published a research paper concerning the design and fabrication of the microfluidic chips and experimental results for the on-chip crystallization of soluble proteins with the dialysis method. The paper entitled ´´A microfluidic device for both on-chip dialysis protein crystallization and in situ X-ray diffraction´´ (Lab Chip, 2020) may be found here: https://doi.org/10.1039/C9LC00651F (it’s an open access article).

Did the ITN help you in the implementation of your project until now?

The answer is definitely yes! I received a lot of help from our collaborator Jean-Baptiste Salmon (LOF, UMR 5258 CNRS-Solvay-Université de Bordeaux). I spent two months in Bordeaux during the first year and two more weeks during the second year of my thesis. There, I learned everything I needed to know about microfluidics and microfabrication.

I should also mention that the other students of the RAMP network, during our common workshops, helped me to understand concepts or methods related to membrane proteins. They were always very patient and willing to explain me various stuff, and for that I thank them all. And I want to thank again Jannik Strauss and Samuel Hjorth-Jensen not only for providing me samples with their protein, but mainly for showing me that with their contribution they really believe and support my project in microfluidics.

Would you recommend other students to apply to a position within a MSCA network such as RAMP? What advice would you give them?

Yes, I would strongly recommend that. Being a member of a MSCA network can provide additional experience and opportunities in contrast to other doctoral programs. What I really like about our network is the diversity of members’ scientific background. I am an engineer, but I am collaborating with structural biologists or crystallographers towards a common goal.

But MSCA networks provide more than scientific opportunities. I am learning how to better communicate with people of various backgrounds, how to make new acquaintances and build my network. And last but not least, during my secondments within the RAMP network, I learned how to adapt to new conditions faster and more efficiently.