Circularized nanodiscs for structural studies of membrane proteins
|Main area:||Protein chemistry|
Although membrane proteins (MP) perform multiple important functions in the organism and are targets for ~60% of current drugs, structures are scarce compared to soluble proteins. This is primarily a result of the fact that MPs are notoriously difficult to handle due to their partly hydrophobic and partly hydrophilic nature. Many different systems are developed with the aim of keeping MPs in solution in a functional state, however, all of the methods have drawbacks.
In our group, we are developing the interesting nanodisc technology. The nanodisc consists of two membrane scaffold proteins (MSP) surrounding a lipid bilayer to form a disc. The technology has proven very useful for functional studies, since the lipid bilayer provides a more native like environment compared to for instance detergents. Recently, “circularized” nanodiscs, i.e. the MSP N- and C- termini covalently linked together by the Sortase enzyme, were developed and shown to have higher thermal stability and a more well-defined size. We are continuing on this development to produce constructs that suit our MPs the best, and at the same time optimizing protocols for expression and purification.
We are currently investigating a number of interesting membrane proteins including Tissue Factor and CorA. Tissue Factor plays an important role in the human blood coagulation pathway, where it interacts with Factor VIIa on the surface on the cell membrane and initiates the coagulation. CorA is a bacterial metal ion transporter, which undergo a major structural rearrangement upon binding of magnesium. Due to the different sizes of the transmembrane domains and due to different experimental requirements, different nanodisc diameters are needed to obtain optimal samples and data. Thus, the end goal is to establish an optimal platform for structural studies by SAXS, SANS, NMR and Cryo-EM.
The experimental work will mainly be conducted in the group of Lise Arleth. The lab is very well equipped with biophysical instruments, and there is easy access to specialised equipment at collaborators at the HCØ institute. You will be working in a multidisciplinary environment among PhD students and post docs with backgrounds in physics, nanoscience and biochemistry.
The project is meant to be a master’s thesis, and previous work (from BA project e.g.) with protein expression and purification is a requirement.
Overview of the Sortase reaction forming the circularized nanodisc. 1) N-terminal His-tag is cleaved off by TEV protease yielding a N-terminal Glycine. 2) Sortase links the N-terminal Glycine with the LPETG-motif to produce a circularized MSP. 3) Nanodisc is formed by mixing MSP and lipids.
The student conducting this project will:
If you are interested or you have any questions, please do not hesitate to contact:
|Supervisor(s):||Lise Arleth (NBI) and Birthe Kragelund (SBiN-lab)|