Knowledge of energetic contributions to protein folding is essential for many of the central quests of structural biology, including protein design and modeling, as well as an understanding of protein function and dysfunction in disease. Consequently, an enormous effort has been made to work out the forces that stabilize protein structure. In this effort, membrane proteins have been largely neglected, in spite of the fact that they play critical biological roles and are the major targets of drugs. Membrane protein folding has been avoided because of the enormous complexity of the membrane environment and the technical challenges faced when studying this problem. In the prior grant period, we have overcome many of these challenges and are finally well positioned to dissect the energetics of folding in a large membrane protein. To my knowledge, there is no other group that combines both thermodynamic stability measurements and detailed structural analysis in a similar way. The Specific Aims are: I.To probe packing forces, we will introduce bumps and create holes at various points in the bacteriorhodopsin (bR) structure and examine the consequences for bR structure and stability. II. To probe hydrogen bond strength, we will specifically delete inter-helical hydrogen bonds in bR and examine the consequences for bR structure and stability. III. To probe the contribution of interhelical loops, we will examine the consequences of side chain deletions and loop insertions on bR structure and stability. IV. Develop hydrogen exchange as a means to probe the unfolded state of bR.