| Abstract
| - 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- andlong-chain phosphatidylcholine mixture to dilute BR within the lipid bilayer. The fraction of BR in thelattice form as a function of added lipid was determined by visible circular dichroism spectroscopy andfit with a cooperative self-assembly model to obtain a critical concentration for lattice assembly. Criticalconcentration values of wild-type and mutant proteins were used to calculate the change in lattice stabilityupon mutation (ΔΔG). By using this method, a series of mutant proteins was examined in which residuesat the BR−BR interface were replaced with smaller amino acids, either Ala or Gly. Most of the mutantlattices were destabilized, with ΔΔG values of 0.2−1.1 kcal/mol at 30 °C, consistent with favorablepacking of apolar residues in the membrane. One mutant, I45A, was stabilized by ∼1.0 kcal/mol, possiblydue to increased lipid entropy. The ΔΔG values agreed well with previous in vivo measurements, exceptin the case of I45A. The ability to measure the change in stability of mutant protein complexes in a lipidbilayer may provide a means of determining the contributions of specific protein−protein and protein−lipid interactions to membrane protein structure.
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