Biophysical studies of lipid-protein interactions

Project Outline

Knowledge of the structure of a cell membrane and the interactions between its components is crucial to understanding membrane properties and function. It is established that bacterial cell membranes differ in lipid composition from eukaryotic plasma cell membranes, as do different membranes within cells. Little is known about the reasons for this diversity, although some specific interactions between lipids and proteins have been identified. Our projects focus on the interactions between lipids or between lipids and proteins, using a range of structural and spectroscopic tools.

Using combined electrochemical and structural/spectroscopic measurements for a range of lipid compositions, we will be able to establish the roles of surface charge vs specific lipid headgroup structure in the interaction of small cationic peptides with model mammalian and bacterial cell membranes. This will enable us to understand whether there are specific interactions that can be targeted for designing new antimicrobial agents or anti-tumour drugs that are selective toward the often negatively charged membranes in these cells. These studies have the potential to be used to screen potential target compounds to combat infection in humans, animals or plants and so would find ultimate potential application in protecting crops, animals, people or reducing the number of compounds tested on animals.

Other projects focus on the interactions of signalling lipids with other components of the membrane. A range of physical techniques, such as atomic force microscopy, neutron and x-ray reflectivity and vibrational spectroscopy will be employed to characterise how glycosylated lipids that activate invariant natural killer T-cells (iNKT) interact with different parts of the mammalian cell membrane and how the membrane is affected by the incorporation of the glycosylated lipids. The extraction of these lipids from the membrane by saposins will be studied at a molecular level.

The study of intrinsic membrane proteins and their interactions with membrane lipids has remained frustratingly challenging, yet knowledge of their structure and interactions is fundamental to our understanding of biology, a fact that is mirrored by the observation that more than 50% of therapeutics and 70% of agrochemicals target membrane proteins. One of the most important issues in studying membrane proteins is the process of extracting them from membranes in a form that is pure, active and stable. We have developed a method that extracts a range of stabilised membrane proteins, using a polymer based on Styrene Maleic Acid (SMA) that excises a 10 nm diameter disc (SMALP) from cell membranes to produce a particle that contains a membrane protein in its local lipid environment. However, our understanding of the processes that underlie the formation of these particles is still limited. In this project we shall use a range of biophysical techniques to determine the kinetics of the process from model membranes and the nature of the interaction between the polymers and the lipids. We shall study the processes with a range of compositions typical of mammalian, bacterial, plant and fungal membranes to ascertain whether there are specific interactions that can be exploited for targeted use of the polymers. We shall then employ similar strategies to study the interaction of the polymer with membrane proteins and smaller peptides, to enable us to predict whether a protein is likely to retain its function within a SMALP. Taken together these studies will provide insights that will help to expand the use of SMALP technology in a wide range of commercial and academic applications.

Techniques

• Protein production through microbial expression
• Membrane protein purification
• Polymer synthesis and modification
• Biophysical measurements including:
  • Surface pressure-area isotherm measurements and monolayer transfer
  • Neutron and X-ray Reflectometry, Grazing Incidence X-ray Diffraction
  • Neutron and X-ray Scattering
  • Capacitance and impedance measurements
  • Atomic Force Microscopy
  • Interfacial vibrational spectroscopy (infrared and Raman)
  • Circular Dichroism spectroscopy
  • Fluorescence spectroscopy
  • Fluorescence microscopy
  • NMR
  • Light scattering
• Collaboration with synthesis groups

There is also potential to visit a collaborator in Germany working on molecular dynamics simulations and to learn these techniques.