From Sequence to Structure- Characterizing Human and Plant Aquaporins

Abstract: Aquaporins constitute a class of membrane proteins that create pores through the lipid bilayer of biological membranes, facilitating the passage of water and other small solutes. Nearly all living organisms possess a more or less intricate set up of aquaporin proteins and the importance of these channels has been implicated in many aspects of health and disease. The aim of the projects summarized in this thesis was to increase the knowledge about the structure and function of aquaporins. A method for obtaining high levels of recombinantly expressed membrane proteins in the yeast Pichia pastoris by screening for clones with multiple recombinant gene inserts was developed. Subsequent to overexpression, a number of different purification schemes for obtaining preparations for molecular characterization were established and three aquaporin isoforms; human HsAQP5 and HsAQP8 and a plant aquaporin, NbXIP1;1, were successfully purified. The two human isoforms were reconstituted into artificial liposomes and by functionality assessments their water transporting capacity was confirmed. The verification of retained functionality after recombinant expression and subsequent purification trials is crucial in order to validate the quality of the protein that goes into crystallization and structure determination trials. By using two different crystallization techniques, diffracting crystals of both HsAQP5 and HsAQP8 were obtained and subsequently, a high resolution structure of HsAQP5 was solved. A different approach was used to identify hypothetical aquaporins in the chloroplast, which is the organelle responsible for the conversion of sunlight into chemical energy in plants. By immunoblotting procedures and mass spectrometry analyses, the presence of several aquaporin isoforms was confirmed, suggesting a possible dependence of the photosynthetic reactions on an assisted trans-membrane water flux.