Thermodynamic stability of the bacteriorhodopsin lattice as measured by lipid dilution

To determine the strength of noncovalent interactions that stabilize a memb rane protein complex, we have developed an in vitro method for quantifying the dissociation of the bacteriorhodopsin (BR) lattice, a naturally occurri ng two-dimensional crystal. A lattice suspension was titrated with a short- and long-chain phosphatidylcholine mixture to dilute BR within the lipid b ilayer. 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 c ooperative self-assembly model to obtain a critical concentration for latti ce assembly. Critical concentration values of wild-type and mutant proteins were used to calculate the change in lattice stability upon mutation (Delt a DeltaG). By using this method, a series of mutant proteins was examined i n which residues at the BR-BR interface were replaced with smaller amino ac ids, either Ala or Gly. Most of the mutant lattices were destabilized, with Delta DeltaG values of 0.2-1.1 kcal/mol at 30 degreesC, consistent with fa vorable packing of apolar residues in the membrane. One mutant, I45A, was s tabilized by similar to1.0 kcal/mol, possibly due to increased lipid entrop y. The Delta DeltaG 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 deter mining the contributions of specific protein-protein and protein-lipid inte ractions to membrane protein structure.