Analyses of complete genome sequences indicate that over 25% of an organism’s proteins are embedded in cellular membranes. Transmembrane proteins play vital roles in cell-cell communications, transmembrane signaling, ion transport and maintenance of cell structure and are the targets for the majority of pharmaceuticals in use today. In addition, the misfolding of specific transmembrane proteins can result in disease, such as in cystic fibrosis. In spite of the vast importance of transmembrane proteins, there are far fewer structures and molecular mechanisms known for transmembrane proteins than for soluble proteins. This difference is due to the presence of hydrophobic sequences that can make it difficult to express and isolate large amounts of these proteins and makes them refractory to many biochemical and structural methods. We are taking a two-pronged approach to improve our understanding of transmembrane proteins: structure/function studies of several specific proteins and a proteomics-level approach to improving methods for studying transmembrane proteins.
Mutations in some transmembrane proteins cause genetic disease.
Mutations in the CFTR cause the genetic disease cystic fibrosis.
Molecular mechanisms of multidrug resistance proteins. Multidrug resistance, a serious and increasingly common medical problem, is due to the action of transmembrane transporters that expel a broad range of molecules, including antibiotics and anti-cancer drugs, from pathogens or cancer cells. Structures of the transporters are needed to elucidate mechanisms and develop possible countermeasures. We are studying two multidrug resistance transporters from bacteria.
Schematic diagram of transmembrane protein topology.
Proteins vary in the number of transmembrane helices and in the number and size of N-terminal, C-terminal, and interhelical domains.
Proteomics-level approach to TM protein studies. Our second approach addresses the need for improved methods for studying transmembrane proteins. We are using a systematic proteomics study to test the expression and membrane localization of over 100 transmembrane proteins that vary in several characteristics (number of transmembrane helices, function, etc.). By identifying which proteins are overexpressed and correctly localized to the membrane under various growth conditions, we hope to determine if expression levels correlate with physical characteristics or functions of the proteins. This project lays the groundwork for future projects to develop improved methods for expression (strains, temperatures, etc.), solubilization, and purification, and characterization of transmembrane proteins.
Inclusion body formation.
Recombinant expression of transmembrane proteins can result in the transmembrane protein being found unfolded or misfolded in insoluble inclusion bodies instead of being correctly targeted and folded in the membrane.