Final Report Summary - PEPTIDE PARTITIONING (Experimental measurement of the direct partitioning of peptides into lipid bilayers) The transfer of peptide segments into the lipid bilayer to form stable transmembrane (TM) helices is the crucial first step in membrane protein folding and assembly. However, the mechanisms that drive this process are not fully understood. A recent experimental assay has measured the insertion of designed peptide sequences into the endoplasmic reticulum membrane via the cellular translocon machinery. This has provided the first quantitative estimate of the translocon to membrane transfer free-energy of polypeptide segments. However, no suitable experimental setup currently exists that allows the direct measurement of the free energy change involved in transferring peptides from water into lipid bilayers. This is because peptides that are hydrophobic enough to insert into membranes generally aggregate in solution. Over the course of this project we have developed a combined computational - experimental technique that allow direct quantification of water to bilayer transfer free energies and applied it to a systematically designed a set of synthetic membrane associated peptides. Using unbiased atomic detail molecular dynamics simulations we were able to fold single TM helices directly into membranes. This in silico approach starts with a non-native protein configuration, usually a completely extended polypeptide, in water. The protein is then allowed to fold freely, as well as partition spontaneously into and out of lipid bilayers. No restraints or biasing potentials are required. Simulation timescales of 1-2 microseconds are usually sufficient to find the native state and fully sample all thermally accessible states. The equilibrium configurations were verified using synchrotron radiation circular dichroism spectroscopy, as well as an experimental membrane insertion assay that utilises the cellular translocon machinery to insert TM segments. In addition, we have developed a purely experimental setup that directly measures the water to bilayer partitioning properties of a series of systematically designed peptides that are water soluble. These were derived by re-engineering of a peptide called pH (low) insertion peptide (pHLIP), which is based on helix C of bacteriorhodopsin. The peptides have a set of unique properties. They are soluble at neutral pH and spontaneously insert into lipid bilayers when the pH is lowered. The peptides are monomeric both in solution at neutral pH and in the bilayer at low pH, allowing direct determination of their partitioning free energy. Direct measurements of peptide partitioning free energies are highly desirable for ab initio membrane protein structure determination, as well as the design of synthetic membrane proteins. The approaches pioneered in the present study have demonstrated the feasibility of using experimentally validated unbiased long timescale atomic detail molecular dynamic simulations to fold TM segments into their free energy minimum. Thermodynamic quantities derived from these simulations were found to be in excellent agreement with the experimental measurement, suggesting that future developments will be able to generalise this approach to predict the fold of more complex multi-span membrane proteins. This promises to greatly improve our understanding of how membrane proteins are assembled in the bilayer, as well as permit the systematic design of novel membrane protein folds.