In biological systems, proteins catalyze the fundamental reactions that underlie all cellular functions, including metabolic processes and cell survival and death pathways. These biochemical reactions are rarely accomplished alone. Rather, they involve a concerted effect from many proteins that may operate in a directed signaling pathway and/or may physically associate in a complex to achieve a specific enzymatic activity. Therefore, defining the composition and regulation of protein complexes is critical for understanding cellular functions.

To understand membrane protein structure and evolution, the molecular interactions that generate protein function, and how these functions are altered or impaired in disease states, it is essential to explore the molecular forces that operate in a membrane environment. An experimental approach for probing the energetics of molecular interactions in a protein is to introduce mutations that add or delete interactions and then assess the effect of the mutation on structure and thermodynamic stability. Soluble proteins have been probed extensively by mutagenesis experiments and these results have led to a much better understanding of the factors that stabilize their structures. The bilayer is a completely different environment, however, so the enormous data available for soluble proteins have only limited applicability to proteins in the membrane. The only membrane protein of known structure that has been extensively examined in a similar way is the simple dimeric transmembrane helix of glycophorin A, which represents only a single helix packing motif. Analogous experiments have not been performed on more complex polytopic membrane proteins, because the confluence of features needed to perform these studies is difficult to achieve. These requirements include a protein that has a known structure so interactions can be identified, that can be obtained in large quantities for physical studies, that unfolds reversibly so thermodynamic stability measurements can be made, and whose sequence can be easily altered. Bacteriorhodopsin (bR), a light driven proton pump from Halobacterium halobium is one of the few, if not the only, helix-bundle membrane protein that satisfies all these requirements. Here we utilize the bR system to examine the consequences of deleting 24 different side-chains on structure and thermodynamic stability.