Design of soluble transmembrane peptides as mimics and inhibitors of membrane protein folding & function.
Published: 2004
Language: eng
Number of pages: 156
Description: Membrane proteins are enticing molecules to study from a structural standpoint because their membrane domains have remarkably uniform architectures---composed of 20--25 residue, canonical alpha-helices. However, as highly hydrophobic molecules, they are equally precarious to study because they pose significant technical challenges involved with their overproduction, purification, handling and subsequent structural analysis. To address this, we developed a method that allowed us to produce large quantities of TM segments through the design and synthesis of so-called "Lys-tagged" TM peptides. Placement of hydrophilic Lys residues at the termini of hydrophobic TM segments rendered peptides completely soluble in water. However, when challenged with a membrane-mimetic environment, these molecules spontaneously inserted and adopted an alpha-helical conformation. Further, these Lys-tagged TM peptides retained their native-like oligomeric states, thus allowing us to readily study the folding and assembly of TM helices. This technology was then applied to the Ff phage major coat protein (MCP) to study residue-specific effects on MCP TM helix-helix interactions; the synthetic approach allowed us to study mutants that were previously unattainable using randomized and saturation mutagenesis protocols on the intact phage. With this enhanced flexibility, a link between the affinity of TM helix-helix interactions and phage viability was described. In a further application of this system, we were able to show that high affinity helix interactive motifs (i.e., GxxxG, and strongly polar residues) were attenuated by their local sequence context. When combined with computer-based simulations and molecular biology approaches, we were able to describe these findings in considerable molecular detail. Finally, we applied the Lys-tagging technology to show that a peptide designed in this way could disrupt the folding of a functional membrane protein. Using the homotrimeric E. coli diacylglycerol kinase (DGK) as a target, we were able to show that a Lys-tagged version of the second TM domain bound to and inhibited DGK function by competing for TM helix packing interfaces. Overall, the work presented here demonstrates the power of the 'domain' approach for providing valuable information regarding the systematic rules which guide the folding and assembly of membrane proteins.
Notes: Adviser:  Charles Deber.

Thesis (Ph.D.)--University of Toronto, 2004.

Electronic version licensed for access by U. of T. users.

Source: Dissertation Abstracts International, Volume: 65-05, Section: B, page: 2296.