MSc Thesis Presentation: Structural studies of human Aquaporin-1 in polymer nanodiscs, and an investigation into a conserved hydrogen-bond network crucial for stability

MSc Candidate 

Philip Drewniak 

Abstract

The human Aquaporin-1 (AQP1) membrane protein was the first water-transporting channel protein discovered, and is integral towards many cellular processes involved with water permeability. Aquaporins (AQPs) are increasingly reported to be quite involved in human disease, especially in some kinds of cancers, such as breast, lung, and colon cancer. This demonstrates the need for further structural studies into AQP1, as protein- protein interactions regulate the processes in which AQP1 is trafficked to the plasma membrane. To do this, a platform that can suspend AQP1 in solution to allow for solution NMR studies must be developed. To this end, the novel reconstitution of the human Aquaporin-1 into polymer nanodiscs was explored, using the new styrene maleic-acid copolymer system which solubilizes membrane proteins directly from cellular membranes. The size and stability of these SMALPs (styrene-maleic acid lipid nanoparticles) were assayed, and chemical shifts possibly belonging to the C-terminal tail of AQP1 was observed in solution NMR. AQP1 has a few potential binding partners in this tail region, which may govern its membrane trafficking and in turn its regulation. None of these potential binding events have ever been physically proven given the absence of a proper platform. This work is the first established nanodisc platform of AQP1 that provides well-resolved solution NMR spectra, and the first step towards future protein-protein interaction studies of this membrane protein.

The second project in this thesis comprised of exploring a highly conserved hydrogen-bond network that exists in stabilizing the overall structure of AQP1. Prior solid-state NMR, structural and conservation analysis revealed the residues involved in this network. Mutagenesis of these sites and the use of attenuated total reflectance (ATR)- FTIR spectroscopy and hydrogen/deuterium exchange with increasing temperatures was used to probe the stability of these new mutants compared to the WT. The mutants studied had varying degrees of overall stability, but some elicited such a drastic change in stability that large differences in even secondary structure were observed. Overall, a unique look into the stabilizing interactions that contribute to AQP1’s overall fold was investigated for the first time.

Examination Committee

• Dr. John Dutcher, Chair 
• Dr. Leonid Brown, Advisor
• Dr. John Dawson, Advisory Committee 
• Dr. Rui Huang, Graduate Faculty