Sofia Trampari’s update on her PhD

SofiaTrampariCut– Could you remind us briefly what your project is about?

My project is about investigating the dynamics and behavior of lipid-detergent-protein micelles. It is a biophysical study on the phase behavior of membrane proteins in the presence of lipids and detergent by a crystallographic and electron microscopy (EM) perspective.

Proteins are fundamental for life and for most of its functions. Membrane proteins are part of the membranes of the cells and between many functions that they have, they can control the flow of nutrients in and out of the cells. Ion transporters such as the Na,K-ATPase generate inward Na+ and outward K+ concentration gradients. Neurotransmitter transporters of the SLC6 family (also referred to as neurotransmitter-sodium symporters, NSS) terminate synaptic signal transmission by Na+ dependent reuptake of released neurotransmitters. Amino acid transporters of the same family also control uptake and homeostasis of amino acid coupled processes in the cell. MhsT is an NSS homologue from Bacillus halodurans, and structures have been determined of occluded, inward-facing states with bound Na+ ions and L-amino acid that provide insight into the cytoplasmic release of Na+ and substrate. MhsT is an excellent model system for structural and functional investigations of human neurotransmitter and amino acid transporters such as dopamine, serotonin, glycine, GABA transporters, B0AT3.

As membrane proteins have such an impact, it is important to study their functions. The most common way to do it is X-ray crystallography. HiLiDe is a systematic crystallization method for membrane protein solubilized in lipid:detergents micelles. Using MhsT as a model system for HiLiDe method the first goal is to create a crystallization phase diagram that can be informative for other membrane proteins. Another goal is to study the phase behavior of the system that is created on this method- lipid-detergent-protein complex. Negative stain and cryo-EM studies are conducted in order to achieve this goal.

During the secondments that this project includes, I did some SEC-SAXS experiments on my proteins to observe the morphology of the complex in different detergent ratios at UHAM. In near future, I will work on a new target using HiLiDe method from an industrial point of view at Novartis. The next stop will be in Grenoble to learn the microfluidic chip technique and last but not least as a collaboration with MDL we will attempt to design commercial screens for the HiLiDe method.

– What important milestone have you reached until now?

As a physicist it was a challenge to work on a biochemistry lab and keep up with the everyday challenges of cell growth and protein production. My first goal was to get familiarized and expertise all these techniques. This was the first important milestone that I succeeded.

Another challenge was the creation of the phase diagram. Large amount of protein have been used and being economical with protein consumption such as finding minimal, reproducible volumes from the pipettes was an obstacle that needed to be overcome. Finally, I completed a diagram and now the next step will be to apply it to more membrane proteins and test its reproducibility for them.

– Did the ITN help you in the implementation of your project until now? If yes in what way?

The workshops that we participated had great impact in my work. It was a great opportunity to be part of them and communicate with so great scientists. I am applying most of this knowledge to my project. Another thing that has great impact on my project is the RAMP community. We are communicating for the problems we may have, we share knowledge and ideas. Secondments and student exchange helps me a lot to the evolution of my work.

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

MSCA is an amazing opportunity to evolve science. Giving you the chance to work with the experts on your field is the best way to get experience and evolve your skills. But MSCA is not just a scientific network. You get the chance to be part of a bigger community that teaches you how to deal with everyday challenges efficiently, travel, work with multibackround people for one goal and exchange ideas.

I highly recommend to apply to a position within a MSCA network and I advise them to live it as much as they can.

 – Is there a topic you would like to share/collaborate within the network in relation to your research work?

In general terms, all the projects that are part of this network are supplementary to each other and we can collaborate so we achieve our goal which is to rationalize membrane proteins. Specifically, currently I am finishing the HiLiDe phase diagram for lipids and detergents. The goal would be to apply it in multiple membrane proteins and check its reproducibility. I would like to test the membrane proteins that the members of my network are working with and hopefully the crystallization of their proteins can help them with their structural studies.

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