FREETH, JAMES,ALEXANDER (2014) The interactions of viral matrix proteins with lipid membranes. Doctoral thesis, Durham University.
|PDF (James Freeth's Thesis) - Accepted Version|
This thesis describes the work undertaken to study the binding of lipid membranes by the viral matrix proteins hRSV-M and Influenza-A-M1.
hRSV-M was recombinantly expressed and purified. It was the subjected to analysis by Langmuir-Blodgett trough experiments, Brewster angle microscopy, Confocal microscopy of giant unilamellar vesicles (GUVs), and binding studies with lipid nanodiscs. These studies showed hRSV-M having a preference for interacting with negatively charged lipids, namely phosphatidylserine, and for having different behaviours in Lo and Ld phases of membranes.
During work on hRSV-M to improve its stability, it was discovered calcium stabilised the protein. This relationship was explored by ICPMS, differential scanning fluorimetry (DSF), circular dichroism (CD), mass spectrometry and microscale thermophoresis. This showed the hRSV-M is a calcium binding protein, containing two binding sites.
Influenza-A-M1 was cloned into a plasmid vector and subsequently expressed and purified. The stability and structure of the protein was probed by DSF and CD measurements. The lipid interactions of this protein were then also explored by Langmuir-Blodgett trough isotherms and GUV binding under confocal microscopy. These showed that M1 is able to bind to phosphatidylserine containing membranes and causes vesicle budding from those membranes.
|Item Type:||Thesis (Doctoral)|
|Award:||Doctor of Philosophy|
|Keywords:||Influenza, hRSV, respiratory syncytial virus, Matrix protein|
|Faculty and Department:||Faculty of Science > Chemistry, Department of|
|Copyright:||Copyright of this thesis is held by the author|
|Deposited On:||30 Oct 2014 11:33|