A quantitative model describing how molecular driving forces balance to shape structure and function in biological membranes is immensely important for both understanding cellular vitality and curing diseases. To accomplish this goal, general principles that report on the unique physical chemistry of protein and lipid interactions must be determined. The lipid bilayer is a chemically diverse and asymmetric structure, and this complexity gives rise to a multitude of factors that influence the energy landscape of integral membrane proteins. In this work, we investigate how depth-dependent changes in local water concentration and the transduction of mechanical forces via the bilayer alter structure and thermodynamics of β-barrel membrane proteins. We determined that backbone hydrogen bonds located in the bilayer interface and those found in soluble portions contribute equally to the folding free energy of the protein. This was achieved using a combination of experimental and theoretical methods, and our findings show that backbone hydrogen bond stabilities in OmpW are similar in magnitude to those found in water soluble proteins. In addition, we developed a theoretical framework describing how transmembrane β-barrel proteins respond to bilayer tension. We then applied this framework to study a total of eight outer membrane proteins from Gram negative bacteria. Our studies revealed that β-barrel membrane proteins are the most rigid component of the cell envelope and strengthen the mechanical properties of the outer membrane in a concentration dependent fashion. This stiffness is observed in all outer membrane proteins investigated, regardless of size, species of origin, or function. Finally, we observe that bilayer tension can remodel β-barrel shape to make these proteins more circular. Protein-protein interactions reduce this reshaping and suggest a general function for complex formation in outer membrane proteins. These studies help to clarify the role of the membrane in influencing protein energetics and motivate future research on the interplay between lipid and proteins in the native environment
