– Could you remind us briefly what your project is about?
I am following novel approaches to solve high resolution structures of membrane-bound pyrophosphatases (mPPase) using a technique called x-ray crystallography (many of us have already talked about that previously, just check out earlier blog posts). For the proteins I am working on, the most commonly used methods for crystallisation and structure solution have failed so far. Therefore, we are currently trying to generate stabilised versions of one mPPase by introducing mutations. In a number of cases, this has been shown to improve the protein expression, simplify the protein handling and purification and/or aid in crystallisation. A second approach we are following to improve the diffraction properties of our crystals is the lipid cubic phase (LCP) crystallisation. This method has been proven useful for the crystallisation of challenging targets such as membrane proteins in many other studies. Only a good diffracting crystal will allow you to solve a protein structure at a high resolution and I am not quite there yet, unfortunately.
– What important milestone have you reached until now?
Until now, I managed to express and purify three mPPases, originating from different species. In one of them, I introduced several mutations and evaluated their effect on the overall protein stability in a so called thermostability assay. This allowed me to identify a set of mutations that renders the protein more stable. After verifying that my mutated protein is still functional, I will start crystallisation trials and hopefully solve its structure by the end of my PhD.
I also managed to get crystals of another mPPase I am working on using both,
the more common vapour-diffusion technique and the uprising LCP method.
However, I still need to optimise the crystals obtained, as they are not good enough
yet to get me to a high-resolution structure.
– Did the ITN help you in the implementation of your project until now? If yes, in what way?
The ITN was very important for the implementation of my project as I was given the opportunity to go on a two-week workshop in Maynooth/Dublin to learn about crystallisation in general and the LCP method (check out my previous blog post). With the acquired knowledge, I was able to get LCP crystals of one of my proteins.
My first secondment at the Imperial College in London was also important for the progress on my work here in Leeds, as I was taught a technique to evaluate the effect of introduced mutations on the stability of my protein of interest.
– Would you recommend other students to apply to a position within a MSCA network such as RAMP?
I can definitely recommend joining a MSCA network! RAMP opened many doors for me and gave me the opportunity to kick off my scientific carrier with a big network of excellent researchers across Europe. The secondments help to extend your network even further and allow you to complement your set of skills. If you want to do a PhD, I recommend to definitely start by looking for MSCA networks that fit your interests first. I am a big fan!