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Dr Alexander Golovanov (MSc, PhD) - postgraduate opportunities

Characterising surface properties of protein biopharmaceutical molecules

Protein biologicals have great potential for the effective treatment of a wide range of diseases. However, aggregation and instability are inherent issues with all protein pharmaceuticals, which is a problem that can limit their utility and developability. Therefore, a better understanding of the molecular basis of protein aggregation, along with the development of formulations to improve protein stability and solubility, would have a major impact on the generation of new therapeutics. Protein aggregation and self-association, as any protein-protein interaction, is primarily mediated by the surface of the molecules. Therefore, understanding the surface properties of proteins (including biopharmaceuticals), and how protein surface interacts with the chemicals used in formulations, is the key for producing improved therapeutics.

 

The aim of this project is to develop new approaches for the characterisation and probing of protein surface properties using solution NMR spectroscopy, and correlate these with theoretical calculations using the 3D structure of these proteins and development of computational algorithms.

 

This multidisciplinary project will be carried out under the supervision of Drs Alexander Golovanov and Jim Warwicker. The student will receive extensive training in modern high-resolution multi-dimensional NMR techniques, protein modelling and computational biology. The Faculty of Life Sciences has state-of-the-art research facilities, including recent investment in the NMR facility that is now equipped with multichannel 500, 600 and 800 MHz spectrometers with cyroprobes. The supervisory team has strong links with industry, which will help to disseminate/ commercialise the discoveries made in this project to the biotechnology and biopharmaceutical sectors.

 

Molecular mechanisms of herpesviral infection.

Herpesviruses are known to cause a wide range of human diseases, ranging from simple cold sores to cancer, Kaposi Sarcoma. These viruses take over the normal cellular mechanisms, for example, mRNA nuclear export, and force cells to produce viruses instead. Understanding these processes at molecular level may help to create new drugs which block virus replication, and thus relieve or prevent such diseases. Kaposi Sarcoma, for example, affects immunocompromised patients and is endemic in Africa, where it is linked with the spread of HIV infection and AIDS.  

 

The student will study how exactly ICP27 and other signature proteins coded by herpes viruses (in the first instance, the common HSV-1) interact with cellular proteins, leading to herpesvirus hijacking human cell. A wide range of biochemical and biophysical methods will be used to address these questions comprehensively. The methods will include molecular biology, protein expression, purification and characterization using biochemical and biophysical techniques (e.g. column chromatography, circular dichroism, SPR, ITC, and NMR spectroscopy). The project will be carried out in the multidisciplinary environment of the Manchester Institute of Biotechnology and supervised by Dr Alexander Golovanov.

 

 

 

  • Tunnicliffe RB, Hautbergue GM, Wilson SA, Kalra P, Golovanov AP. (2014). Competitive and Cooperative Interactions Mediate RNA Transfer from Herpesvirus Saimiri ORF57 to the Mammalian Export Adaptor ALYREF. PLoS Pathog, 10(2), e1003907. DOI:doi:10.1371/journal.ppat.1003907

 

  • Tian, X., Devi-Rao, G., Golovanov, A. & Sandri-Goldin, R (2013). The Interaction of the Cellular Export Adaptor Protein Aly/REF with ICP27 Contributes to the Efficiency of Herpes Simplex Virus 1 mRNA Export. J Virol, 87(13), 7210-7217. DOI:10.1128/JVI.00738-13

 

  • Tunnicliffe RB, Hautbergue GM, Kalra P, Jackson BR, Whitehouse A, Wilson SA, Golovanov AP. (2011). Structural Basis for the Recognition of Cellular mRNA Export Factor REF by Herpes Viral Proteins HSV-1 ICP27 and HVS ORF57. PLoS Pathogens, 7(1), e1001244. DOI:10.1371/journal.ppat.1001244

 

Purification and characterisation of the mitochondrial protein-import chaperones

Mitochondrion is an essential organelle playing a central role in many biological processes. Mitochondrial dysfunction leads to life threatening diseases, including diabetes, stroke, Alzheimer’s, and cancer. The small Tim proteins (e.g. Tim9, Tim10, Tim12 …) of the mitochondrial intermembrane space (IMS) play an essential role during import of mitochondrial membrane proteins. They act as novel protein chaperones preventing aggregation of their substrates (membrane proteins) in the IMS and facilitate their insertion into mitochondrial membranes.  In the absence of the small Tim proteins, they cannot be correctly imported into mitochondrial membrane, and cells die. The importance of the small Tim proteins is also illustrated by the observation that a single Cys mutation in the human deafness-dystonia peptide 1 (DDP1, a member of small Tim protein family) causes Mohr-Tranebjaerg syndrome.

 

Despite the important function of the small Tim proteins, their functional mechanism is not clear, especially for Tim12. The overall aim of this PhD project is to investigate the function and functional mechanism of the small Tim proteins with a focus on Tim12.  The student will study how Tim12 interacts with other small Tim proteins (Tim9 and Tim10), and how it mediates the import of its substrates proteins. A wide range of biochemical and biophysical methods will be used to address these questions comprehensively. The methods will include molecular biology, mitochondrial protein import analysis, protein purification and characterization using biochemical assays and biophysical techniques (e.g. column chromatography, circular dichroism, NMR). The project will be carried out in a multidisciplinary environment of the Manchester Institute of Biotechnology and co-supervised by Dr Lu and Dr Golovanov.

 

  • Lu H, Golovanov AP, Alcock F, Grossmann JG, Allen S, Lian LY, Tokatlidis K. The structural basis of the TIM10 chaperone assembly. J Biol Chem. 279(18):18959-66 (2004).

 

  • Lu H, Allen S, Wardleworth L, Savory P, Tokatlidis K. Functional TIM10 chaperone assembly is redox-regulated in vivo. J Biol Chem. 279: 18952-8 (2004).

 

  • Ivanova E, Lu H. Allosteric and electrostatic protein-protein interactions regulate the assembly of the heterohexameric Tim9-Tim10 complex.  J Biol Chem.  379(3): 609-16 (2008).

 

  • Durigon R, Wang Q, Ceh Pavia E, Grant CM, Lu H. Cytosolic thioredoxin system facilitates the import of mitochondrial small Tim proteins. EMBO Rep. 13(10):916-22 (2012).

 

  • Ceh-Pavia E, Spiller MP, Lu H. Folding and biogenesis of mitochondrial small Tim proteins. Int J Mol Sci. 14(8):16685-705(2014).