This thesis presents three different approaches to enhance crystallization of membrane proteins. The
major part of the thesis presents the establishment of a method to stabilize membrane proteins by
mutagenesis. Although it is fairly straightforward to introduce mutations in proteins using random
mutagenesis, the selection of mutants that are more stable than wild type generally involves exhaustive
screening. Adding detergent stability as a selection parameter to the thermal stability, this project
established a generic high throughput screening method using an Escherichia coli sodium-proton
antiporter NhaA as a model. A screening funnel rapidly eliminated unstable mutants based on their
expression level, octyl glucoside solubility, and elution profile of fluorescent size exclusion
chromatography (FSEC), leaving only several mutants to be subjected to rigorous thermal stability
assay. The screening identified 2 mutants, which exhibited significantly improved thermostability at
the protein’s active pH of 8.5. The preliminary crystal structure of an NhaA dimer complex is also
presented. This structure was determined at a resolution of 3.8 Å. Although biochemical studies have
suggested NhaA exists as a dimer in the lipid bilayer, the only available dimer structure has been a
cryo-EM model at 7 Å published earlier this year. Overall the two structures are very similar although
the position of the β-sheet at the dimer interface differs slightly.
Two minor chapters discuss construct engineering and antibody co-crystallization to enhance
crystallization. Constructs were made for five different P450s involved in biosynthesis of the moulting
hormone ecdysone from Drosophila melanogaster and Bombyx mori. The null expression under
different growth conditions suggests the design requires further optimization. Co-crystallization of
human erythrocyte anion exchanger Band 3 with a Fab fragment of BRAC18 resulted in Fab crystals.
The Fab crystal structure was determined to a resolution of 1.6 Å with the view of aiding Band 3: Fab
structure determination
