Membrane proteins are central players in many if not most cellular processes: cell-cell interaction, signal transduction, nerve conduction, small molecule transport, macromolecular trafficking, etc. A growing number of high-resolution membrane protein structures provide important insights not only into function but also into the general structural constraints imposed by the lipid bilayer. In contrast, almost no information is available concerning how membrane proteins fold in vivo. Mainly, this is because of a lack of suitable assays to follow the folding process. The main objective of this proposal is to develop a broad range of new methods, largely based on chemical-biology approaches combined with protein engineering, to study membrane protein insertion, folding, and assembly in vivo or under in vivo-like conditions. We will aim for quantitative studies whenever possible. Questions we will address include: What are the in vivo kinetics of transmembrane-helix integration? What are the energetics of membrane insertion of non-natural amino acid side chains with physico-chemical properties distinct from those of the 20 natural amino acids? What kinds of residue-residue interactions drive interactions between transmembrane helices and between membrane protein subunits? How should we best design and verify novel interacting transmembrane helices? Given the importance of membrane proteins in both basic and applied biological research, we expect that a deeper understanding of the molecular interactions that drive their folding and stabilize their structure in vivo will have a major impact across many areas of molecular life science.