Quenching data for wild-type KcsA were analyzed by using the parameters for binding at the nonannular sites determined above to obtain relative binding constants at the annular lipid binding sites.
Uncharged analogues of BrSA such as 9,10-dibromostearoyl alcohol and 9,10-dibromomethylstearate show very weak binding at the nonannular sites on KcsA.
However, each Trp-67 residue is close to a nonannular lipid binding site, and binding of a brominated molecule results in very efficient quenching, allowing the determination of binding constants at the nonannular sites.
The W67,68 mutant was used to determine binding constants at the nonannular sites on KcsA.
Addition of 9,10-dibromomethylstearate or 9,10-dibromostearoyl alcohol resulted in only low levels of quenching of the fluorescence of W67,68, showing very weak binding to the nonannular sites on KcsA.
Addition of BrSA to W67,68 reconstituted with DOPC resulted in a marked decrease in fluorescence intensity, demonstrating binding of fatty acids at the nonannular sites.
The scaling factor accounts for the fact that in the competitive binding model the nonannular site will be fully occupied by lipid B at a mole fraction xB of 1, whereas this will not be true in the simple binding model where the site might be only partially filled, depending on the value of K2.
We have shown that relative lipid binding constants at annular and nonannular sites on KcsA can be determined using fluorescence quenching approaches.
As a consequence, Trp fluorescence quenching by brominated fatty acids will report just on binding to annular and nonannular sites.
Quenching studies with the KcsA mutant W67,68 show that BrSA can also bind to the nonannular sites on KcsA located one at each protein protein interface in the homotetrameric structure.
