Interactions of transmembrane peptides and proteins with lipid membranes studied by mass spectrometry

The exact way in which membrane proteins are embedded in the lipid bilayer is not well understood. Only limited insight exists in the interactions of membrane proteins with the surrounding lipids and in the membrane-water interface in particular, where many interactions of membrane proteins with substrates, enzymes and ligands take place. Insight into the exact positioning of the transmembrane segments of such proteins with respect to this interface, as well as into their structural properties, are essential to understand the functioning of these proteins. An accepted way to systematically study integral membrane proteins and their interactions with the surrounding lipid membrane, is to use designed model transmembrane peptides in well-defined vesicular systems. These hydrophobic peptides are about as long as a typical transmembrane segment of such a protein and form ? -helices in lipid bilayers. In Chapters 2, 3 and 4 experiments are described using such peptides in order to study specific characteristics of these peptides in a lipid environment. Extending on this, in Chapter 5 a full-length naturally occurring integral membrane protein embedded in a lipid environment is studied. The scope of this thesis is to investigate the possibilities of mass spectrometry (MS) for studying these membrane peptides and proteins in membrane systems. In Chapter 2, it will be shown that it is possible to study the precise positioning of transmembrane peptides in lipid bilayers. Therefore, a novel method was developed to implement MS in membrane peptide and protein research, referred to as proteoliposome spraying method. By combining hydrogen/deuterium (H/D) exchange and nano-flow electrospray ionization (ESI) MS, the numbers of incorporated deuterium atoms in designed transmembrane peptides that are incorporated in lipid bilayer membranes can be analyzed. This proteoliposome spraying method involves direct introduction of the vesicle suspension into the MS-inlet. Several distinct H/D exchange rates can be observed for the exchangeable hydrogens depending on their position in the transmembrane peptides. Assignments are confirmed by collision induced dissociation (CID) MS measurements, which allow analysis of exchange of individual peptide amide linkages. In Chapter 3, this method is used to get more detailed insights into the architecture of various transmembrane peptides and into the positioning of such peptides in lipid bilayer membranes. Several variables such as (1) the influence of the length of the peptide hydrophobic core sequence, (2) the role of the flanking tryptophan residues, and (3) the influence of putative helix breakers, are investigated. The results are discussed in a biochemical and biophysical context. Chapter 4 highlights the possible complications in H/D exchange studies that might result from gas-phase migration of deuterium atoms incorporated in the peptides (referred to as scrambling). Systematic studies of this phenomenon suggest that the extent of deuterium scrambling is strongly influenced by experimental factors, such as the exact amino acid sequence of the peptide, the nature of the charge carrier and, therefore, most likely by the gas-phase structure of the peptide ion. In Chapter 5, an integral membrane protein is explored: the potassium channel KcsA. The intention was to extend the studies presented in Chapters 2 and 3 to a larger membrane protein and to investigate the interactions of this protein with lipids. In these experiments, non-covalent complexes of phospholipid molecules and KcsA are observed. Moreover, it is possible to probe preferential binding of certain classes of lipids to the protein. The results are discussed in the context of results of biochemical assays with identical protein-membrane systems. In Chapter 6, the results presented in this thesis are summarized and discussed in the context of the present literature.