Structural studies of human Aquaporin-1 in polymer nanodiscs and an investigation into a conserved hydrogen-bond network crucial for stability
The human Aquaporin-1 (AQP1) membrane protein is integral towards many cellular processes involved with water permeability. This thesis explores two separate projects towards the further structural characterization of AQP1. Firstly, the novel reconstitution of AQP1 into SMALPs (styrene-maleic acid lipid nanoparticles) was developed and further assayed for stability and amenability towards solution nuclear magnetic resonance (NMR) experiments. A considerable amount of the most mobile amino acids in AQP1 were observed and resolved using this membrane mimetic system. Secondly, this thesis reports an additional focus into a highly conserved hydrogen-bond network existing within AQP1. This network was explored using site-directed mutagenesis and Fourier-transform infrared (FTIR) spectroscopy coupled with hydrogen- deuterium exchange upon increasing temperatures. The mutants studied affected the stability of AQP1 to varying degrees, revealing the importance of these residues towards the overall stability of the native protein fold.