Abstract 3D crystallisation of membrane proteins presents a bottleneck for the determination of the structures of membrane proteins. Obtaining 3D crystals of membrane proteins is made difficult by a number of factors including the poor solubility and instability of membrane proteins outside of their native membrane environment. 2D crystallisation of membrane proteins offers an alternative to preserve the conformational structure and functional activities of membrane proteins within their native bilayer membranes in 2D arrays from which the structure of membrane proteins can be determined. Different techniques exist for obtaining 2D crystals of membrane proteins including surface crystallisation or more commonly 2D crystallisation by detergent removal (using either dilution, dialysis, hydrophobic resin adsorption or cyclodextrin complexation) to promote reconstitution of the protein molecules within bilayer-forming lipids. Another method which has been emerged and is being used increasingly is the lipid monolayer technique for 2D crystallisation of proteins. The use of lipid monolayers to bind and adsorb proteins is an attractive and increasingly important method for generating high localised concentrations of oriented proteins and protein complexes. These bound proteins can be imaged directly, or they may form 2D crystalline arrays that are amenable to structure determination by single particle analysis or 2D electron crystallography. 2D crystals grown by this technique can also be used to initiate the growth of 3D crystals for X-ray diffraction analysis. Many derivatised lipids have been prepared for use with this technique, incorporating a diverse range of ligands to enable binding to specific proteins. Synthetic lipids containing functionalised head groups that chelate Ni2+ or Cu2+ have also been prepared to bind and orient expressed proteins that contain His-tags. Protein-binding monolayer-forming lipids generally consist of two distinct components: (1) a branched hydrocarbon tail to confer fluidity to the monolayer and (2) a functionalised hydrophilic head group to facilitate binding of protein molecules at the air-water interface. Newer examples of these compounds also incorporate perfluorinated hydrocarbon moieties to confer detergent resistance to these lipids. The present work discusses the chemistry of all these functionalised lipids and their contributions to monolayer 2D protein crystallisation. This thesis focuses on the synthesis of novel nickel-chelating fluorinated lipids to be used as a template for 2D crystallisation of His-tagged membrane proteins at the air/water interface. These monolayer-forming lipids have been designed with three distinct components: (i) a branched hydrocarbon tail to confer fluidity of the monolayer, (ii) a perfluorinated central core for detergent resistance, and (iii) a nickel-chelating hydrophilic head group to facilitate binding of recombinant, polyhistidine-tagged fusion proteins. Alkylations of fluorinated alcohols used in these syntheses proceed in good yields only with the application of prolonged sonication and, in some cases, in the presence of phase-transfer catalysts. Biophysical properties of Langmuir monolayers formed by our target synthetic fluorinated lipids were studied, comparing the results obtained with those of DOPC and DOGS Ni-NTA as examples of non fluorinated lipids. The Langmuir films were characterised by surface pressure-area isotherms and X-ray reflectometry to show their fluidity, thickness and packing density. The stability of fluorinated lipid monolayers and their ability to resist the solubilisation effects of a number of detergents were investigated using monolayer and affinity grid techniques. Results showed that fluorinated lipids offer an improved resistance to the solubilisation effects of detergents compared with their non-fluorinated counterparts. A number of trials for 2D crystallisation of both soluble and membrane proteins have been performed using fluorinated lipid monolayers. These new synthetic fluorinated lipids were successfully used to obtain 2D crystals of the His-tagged membrane protein BmrA from Bacillus subtillis by the monolayer technique.
| Identifer | oai:union.ndltd.org:ADTP/286090 |
| Creators | Waleed Hussein |
| Source Sets | Australiasian Digital Theses Program |
| Detected Language | English |
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