A MECHANISM TO ALTER REVERSIBLY THE OLIGOMERIC STATE OF A MEMBRANE-BOUND PROTEIN DEMONSTRATED WITH ESCHERICHIA-COLI EIIMTL IN SOLUTION

This paper reports that the aggregation state of a membrane protein ca n be changed reversibly without the use of chaotropic agents or denatu rants by altering the attractive interactions between micelles of poly ethylene glycol-based detergents, This has been documented using manni tol permease of Escherichia coli (EIImtl), a protein whose activity is dependent on the dimerization of its membrane-embedded domains, We sh ow that the driving force for the hydrophobic interactions responsible for the dimerization can be decreased by bringing the protein into a less polar environment, This can be done simply and reversibly by incr easing the micelle cluster size of the solubilizing detergent since th e micropolarity in the micelle decreases upon clustering and is direct ly related to the cluster size. The micelle cluster size was varied at a fixed temperature by adding sodium phosphate or a second detergent with a distinct clustering behavior, and the changes were quantified b y quasi elastic light scattering and by determining the cloud point or demixing temperature (T-d) of the detergent, Maximal EIImtl activity was found when no micelle clustering occurred, but the activity gradua lly decreased down to 5% of the maximal activity with increasing clust er size, The inactivation was found to be completely reversible, The k inetics of heterodimer formation were also significantly affected by c hanges in the micelle cluster size as expected, Increasing the cluster size resulted in faster formation of functional heterodimers by incre asing the rate of homodimer dissociation. This phenomenon should be ge nerally applicable to controlling the oligomeric state of membrane-bou nd proteins or even water-soluble proteins if their subunit associatio n is dominated by hydrophobic forces.