Thermodynamic stability of the bacteriorhodopsin lattice as measured by lipid dilution.

To determine the strength of noncovalent interactions that stabilize a membrane protein complex, we have developed an in vitro method for quantifying the dissociation of the bacteriorhodopsin (BR) lattice, a naturally occurring two-dimensional crystal. A lattice suspension was titrated with a short- and long-chain phosphatidylcholine mixture to dilute BR within the lipid bilayer. The fraction of BR in the lattice form as a function of added lipid was determined by visible circular dichroism spectroscopy and fit with a cooperative self-assembly model to obtain a critical concentration for lattice assembly. Critical concentration values of wild-type and mutant proteins were used to calculate the change in lattice stability upon mutation (¢¢G). By using this method, a series of mutant proteins was examined in which residues at the BR-BR interface were replaced with smaller amino acids, either Ala or Gly. Most of the mutant lattices were destabilized, with ¢¢G values of 0.2-1.1 kcal/mol at 30 °C, consistent with favorable packing of apolar residues in the membrane. One mutant, I45A, was stabilized by 1.0 kcal/mol, possibly due to increased lipid entropy. The ¢¢G values agreed well with previous in vivo measurements, except in the case of I45A. The ability to measure the change in stability of mutant protein complexes in a lipid bilayer may provide a means of determining the contributions of specific protein-protein and protein- lipid interactions to membrane protein structure.