Thermodynamic measurements of the contributions of helix-connecting loops and of retinal to the stability of bacteriorhodopsin

Thermodynamic studies of bacteriorhodopsin (BR) have been undertaken in order to investigate the factors that stabilize the structure of a membrane protein. The stability of the native, intact protein was compared to that of protein with retinal removed, and/or cleaved in one or two of the loops connecting the transmembrane helices. The stability was assessed using differential scanning calorimetry and thermal denaturation curves obtained from ultraviolet circular dichroism and absorption spectroscopy. Retinal binding and the loop connections were each found to make a small contribution to stability, and even a sample that was cleaved twice as well as bleached to remove retinal denatured well above room temperature. Removal of retinal destabilized the protein more than cleaving once, and about as much as cleaving twice. Retinal binding and the connections in the loops were found to stabilize BR in independent ways. Cleavage of the molecule into fragments did not reduce the intermolecular cooperativity of the denaturation. Dilution of the protein by addition of excess lipid in order to eliminate the purple membrane crystal lattice also did not alter the cooperativity. These results are used to compare the relative importance of various contributors to the stability of BR. Because parts of integral membrane proteins must fold in the hydrophobic region of lipid bilayers, the balance of interactions stabilizing the proteins' structure is likely to be both quantitatively and qualitatively different from that found in soluble proteins. The factors contributing to the stability of proteins that contain several transbilayer a-helices can be separated conceptually into those stabilizing the helices themselves and those stabilizing the interactions between the helices in the tertiary structure (Popot & Engelman, 1990). It has been proposed that the stability of the helices in bilayers arises from main-chain hydrogen bonding and from the hydrophobic nature of the side chains (Engelman et al., 1986) and experiments have shown that some individual helices from a polytopic membrane protein can be considered to be independent folding domains (Kahn & Engelman, 1992; Hunt et al., 1991). The relative importance of the factors leading to the association of the helices has not been as thoroughly explored. Possible major factors stabilizing helix-helix association in bacteriorhodopsin (BR)' can be divided roughly into four categories. First, the extramembranous loops connecting the helices may constrain the positions of the helices by being short or by forming secondary and tertiary structures. Second, polar amino acid side chains within the membrane may cause the helices to associate in such a way as to allow the formation of hydrogen bonds and ion pairs in order to minimize the