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Novel biosensors of protein assembly in and near lipid-bilayers: synthesis and applications

We wish to prepare novel biosensors to detect and quantify protein assembly in lipid membranes. This tool will allow us to answer the following questions:

  1. What is the contribution of lipid composition, protein structure and physical properties of the lipid membrane to the process of protein assembly?
  2. How these factors drive cell signalling and sensing?

Background

The assembly of membrane embedded proteins play a key role in cell signalling essential for cell-cell communication in living organisms. Despite its importance, this process is still poorly understood because the development of methods that can detect and quantify the assembly of proteins in lipid environments is challenging. (1) One problem is that transmembrane proteins and lipid environment are difficult to produce via DNA recombinant technology making it difficult to have functional membrane proteins for in vitro studies. Therefore most of the current methodologies are based on analysis of soluble version of membrane proteins (e.g. without the trans-membrane domains) and on cell biology approaches which have limitations, namely the lack of the essential lipid environment and the complexity of natural cell membranes. One approach to study protein assemblies in or near cell membranes is to use minimal systems that provide good model of the lipid bilayer but are less complex than biological membrane. (2) Examples of such systems are liposomes that we and others have used to study receptor-ligand binding and protein assemblies near lipid bilayers. (3, 4)

Project

The aim of the project is to prepare novel biosensors to detect and quantify protein assembly in lipid membranes. These biosensors will be made of liposomes bearing fluorescent lipids that can be attached to proteins. The idea is that the fluorophore will act as reporter, sensor of protein assembly in the lipid bilayer.

The idea is to use immunoglobulins as the protein models to develop the methodology for the proteoliposomes preparation. The in-membrane assembly of immunoglobulins initiate cell adhesion leading to immunoresponse but the mechanism is still poorly understood. (5) We will study the assembly of the immunoglobulins and the resulting adhesion of the liposomes as a simplify model of cell adhesion.

The proteoliposomes will also be used for the insertion of the fluorescent lipid-immunoglobulin constructs in cell membranes to study the assembly of these proteins and adhesion in biologically relevant membranes.

The work plan will include: the synthesis of the fluorescent lipids, the synthesis of the immunoglobulins labelled with the fluorescent lipids and preparation of the proteoliposomes. The protocols for the chemistry, liposome preparation and analysis of protein assembly will be based on procedures developed by us previously. (3, 4, 6) Work will cell cultures will be carried out in collaboration with Prof. Mike Philpott team.

Training

You will be trained in a broad range of techniques: procedures of synthetic chemistry (NMR, HPLC, MS), methods for liposomes preparation and purification (extrusion, dialysis, GPC), Uv/Vis, fluorescence spectroscopy (for analysis of protein assembly), fluorescence microscopy and transmission electron microscopy (TEM, to visualise cell and liposome adhesion) and techniques for cell cultures.

Eligibility

Applicants should be Chinese nationals and are required to have at least an upper second class degree and a masters degree in a relevant discipline from a top university anywhere in the world.

International students must provide evidence of proficient English language skills, see our entry requirements page for further information.

Some experience in procedures of organic synthesis and characterisation of organic molecules will be ideal. Interests and experience in protein chemistry and protein analysis would be an additional advantage.

The work with cells culture will require collaboration with cell biologists and the candidate is expected to be willing to work as part of a team as well as independently.

The successful candidate will be trained in project management: the efficient use of time, verbal and oral skills to present data to his/her supervisor, in high impact journals and at international conferences.

Application process

Applications for 2015 entry have now closed. To express your interest in this project for 2016 please contact the project supervisor Dr Lilia Milanesi (l.milanesi@qmul.ac.uk) to express your interest in applying.

If she agrees to take your application forward you will need to complete the Queen Mary online application form.

If you are successful we will give you an offer on the condition that you are given a funding award from the China Scholarship Council. When you have received a conditional offer, apply directly to the China Scholarship Council.

Self-funding students

If you are interested in self-funding please contact Dr Lilia Milanesi by email (l.milanesi@qmul.ac.uk) to discuss your eligibility for this project.

Publications

  1. Wu Y. et al. Nature 2011 475: 510-514
  2. Gruber B. et al., Chem. Eur. J 2013, 19, 438-448
  3. S. Tomas, L. Milanesi Nature Chemistry 2010, 2, 1077-1083
  4. L. Milanesi et al., Proc Natl Acad Sci U S A 2012, 109: 20455-20460
  5. Van der Merwe P. A. et al. Nature Rev Immunol 2011 11: 47-55
  6. L. Milanesi et al., Proc Natl Acad Sci U S A 2012, 109, 19563-19568
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