Characterization of membrane proteins: insights into palmitoylation

Membrane proteins are essential for molecular transport and signaling over membranes. Transport and signaling underlie many cellular functions and as a result, membrane proteins are major target in drug development. A better understanding of membrane protein structure and functioning will raise new opportunities to modulate cells and benefit development of novel drugs. In this thesis, we study membrane proteins that span the membrane via multiple ɑ-helices. These transmembrane helices maintain the structure of membrane proteins by weak helix-helix interactions. Currently, a major limitation in obtaining membrane protein structures is poor stability of these proteins in solution. To study membrane proteins in solution, these proteins have to be extracted from the membrane. Proteins are usually extracted by replacing membrane lipids with solubilizing detergents. Detergent selection for membrane protein solubilization and further downstream experiments is very important, as the detergent must retain the weak helix-helix interactions. Therefore, throughout each chapter in this thesis, we have developed and optimized protocols to maintain the native protein conformation in detergent solution. In summary, in Chapter 2, we developed purification conditions tailored for crystallization of tight-junction protein claudin-3 in complex with an enterotoxin from Clostridium perfringens. In Chapter 3, the development of a novel strategy for membrane protein purification is described. This purification strategy enables membrane proteins to be rapidly purified from mammalian cells whilst maintaining their native conformation. The difficulty to purify membrane proteins also limited the study of post-translational modifications of these proteins. In Chapter 4 we apply the acquired knowledge on membrane protein purification and introduce a novel method to detect membrane protein palmitoylation. Mammalian membrane proteins are often modified via covalent attachment of palmitate to fine-tune their functions. Detection of palmitoylation from four membrane protein classes provides novel insight into the mechanism of membrane-protein palmitoylation

Characterization of membrane proteins: insights into palmitoylation

Membrane proteins are essential for molecular transport and signaling over membranes. Transport and signaling underlie many cellular functions and as a result, membrane proteins are major target in drug development. A better understanding of membrane protein structure and functioning will raise new opportunities to modulate cells and benefit development of novel drugs. In this thesis, we study membrane proteins that span the membrane via multiple ɑ-helices. These transmembrane helices maintain the structure of membrane proteins by weak helix-helix interactions. Currently, a major limitation in obtaining membrane protein structures is poor stability of these proteins in solution. To study membrane proteins in solution, these proteins have to be extracted from the membrane. Proteins are usually extracted by replacing membrane lipids with solubilizing detergents. Detergent selection for membrane protein solubilization and further downstream experiments is very important, as the detergent must retain the weak helix-helix interactions. Therefore, throughout each chapter in this thesis, we have developed and optimized protocols to maintain the native protein conformation in detergent solution. In summary, in Chapter 2, we developed purification conditions tailored for crystallization of tight-junction protein claudin-3 in complex with an enterotoxin from Clostridium perfringens. In Chapter 3, the development of a novel strategy for membrane protein purification is described. This purification strategy enables membrane proteins to be rapidly purified from mammalian cells whilst maintaining their native conformation. The difficulty to purify membrane proteins also limited the study of post-translational modifications of these proteins. In Chapter 4 we apply the acquired knowledge on membrane protein purification and introduce a novel method to detect membrane protein palmitoylation. Mammalian membrane proteins are often modified via covalent attachment of palmitate to fine-tune their functions. Detection of palmitoylation from four membrane protein classes provides novel insight into the mechanism of membrane-protein palmitoylation

Site-specific functionality and tryptophan mimicry of lipidation in tetraspanin CD9

Lipidation of transmembrane proteins regulates many cellular activities, including signal transduction, cell-cell communication, and membrane trafficking. However, how lipidation at different sites in a membrane protein affects structure and function remains elusive. Here, using native mass spectrometry we determined that wild-type human tetraspanins CD9 and CD81 exhibit nonstochastic distributions of bound acyl chains. We revealed CD9 lipidation at its three most frequent lipidated sites suffices for EWI-F binding, while cysteine-to-alanine CD9 mutations markedly reduced binding of EWI-F. EWI-F binding by CD9 was rescued by mutating all or, albeit to a lesser extent, only the three most frequently lipidated sites into tryptophans. These mutations did not affect the nanoscale distribution of CD9 in cell membranes, as shown by super-resolution microscopy using a CD9-specific nanobody. Thus, these data demonstrate site-specific, possibly conformation-dependent, functionality of lipidation in tetraspanin CD9 and identify tryptophan mimicry as a possible biochemical approach to study site-specific transmembrane-protein lipidation