Selective Membrane Transport Systems studied by Solid-State NMR Spectroscopy

Abstract

Membranes border cells from their environment. Thus, they belong to the basic prerequisites for life as we know it. Within cells, membranes are used to establish compartments dedicated to specific tasks such as providing local proximity of components, assembling specialized molecular machinery and confining their numerous functions to salutary consistency. Biological membranes are functionalized by associated proteins which account for a large fraction of all proteins encoded by the genome. Not surprisingly, many of the available drugs act via their interaction with these membrane proteins. Despite their large biological and pharmaceutical importance only very few membrane proteins are understood on the molecular level. Often structure and function of these proteins depend on their lipidic environment. Then, traditional techniques like x-ray diffraction or solution-state NMR are not well suited due to the large molecular size and the inhomogeneous character of membrane-protein systems. Solid-state NMR (ssNMR) spectroscopy on the other hand is a prime technique to investigate structure and dynamics of membrane proteins at atomic resolution even in the presence of a lipid bilayer. This thesis reports on progress made in ssNMR methodology, sample preparation, and data analysis enabling the study of two distinct proteins involved in selective membrane transport. First, ssNMR spectroscopy was used to establish structure-function relations of the chimeric potassium (K+) channel KcsA-Kv1.3 which serves as model system for regulated ion transport across membranes essential for cellular excitability. The results provide detailed insight into structure and dynamics of K+ channel activation and inactivation gating and reveal modulating effects of ligands, ions, and the lipidic environment on the functionally relevant conformations of the K+ channel. Second, functional hydrogels formed by FG-repeat domains of the yeast nucleoporin Nsp1p were characterized by ssNMR. These polypeptides constitute an essential part of the nuclear pore complex controlling all molecular trafficking between nucleus and cytoplasm in eukaryotic cells. The acquired data provide structural details of FG-hydrogels as well as their gelation kinetics and link amyloid-like protein-protein interactions to the selectivity barrier of the nuclear pore complex.