Template-directed assembly of receptor signaling complexes

Abstract

Transmembrane receptors in the signaling pathways of bacterial chemotaxis systems influence cell motility by forming noncovalent complexes with the cytoplasmic signaling proteins to regulate their activity. The requirements for receptor-mediated activation of CheA, the principal kinase of the Escherichia coli chemotaxis signaling pathway, can be demonstrated using self-assembled clusters of a receptor fragment (CF) derived from the cytoplasmic domain of the aspartate receptor, Tar. Histidine-tagged Tar CF can be assembled on the surface of unilamellar vesicles via a lipid containing the Nickel-nitrilotriacetic acid moiety as a headgroup. The stability of such a complex can be controlled by the properties of the template including the size and composition, which can be used, for example, to vary the 2-dimensional concentration of receptor fragments. Surface-assembled CF is also found to serve as a substrate for receptor methylation, which is catalyzed by the receptor transferase. Since neither CheA activation nor CF methylation is observed in comparable samples in the absence of vesicles, it is concluded that surface-templating generates the organization among CF subunits required for biochemical activity.

1 - 45 . (canceled) 
     
     
         46 . An in vitro assembly for use in protein assays, said assembly comprising:
 a lipid membrane including a first lipid component; and a plurality of a first membrane-associated protein component, said first membrane-associated protein component having a binding affinity for said first lipid component and being coupled with said lipid membrane by interaction with said first lipid component to promote a two dimensional organization of said plurality of first membrane-associated protein component that mimics a natural organization of said first membrane-associated protein component on a cell membrane; wherein said first-membrane-associated protein component, when coupled with said lipid membrane, exhibits a greater biological function than the first membrane-associated protein component in an aqueous medium in the absence of the lipid membrane.   
     
     
         47 . The assembly of  claim 46  wherein said membrane further comprises a second lipid component that does not have a binding affinity for said first membrane-associated protein component; and wherein said first and second lipid components are present in relative amounts sufficient to at least partially affect the concentration of said first membrane-associated protein component on said membrane. 
     
     
         48 . The assembly of  claim 47  wherein said first lipid component comprises DOGS-NTA-Ni +2 , and said second lipid component comprises DOPC, said first and second lipid components present in a ratio from about 3:2 to about 1:20. 
     
     
         49 . The assembly of  claim 47  wherein said second lipid component is a substrate for said membrane-associated protein component. 
     
     
         50 . The assembly of  claim 46  wherein said membrane is a unilamellar vesicle. 
     
     
         51 . The assembly of  claim 50  wherein said vesicle has a diameter dimension up to about 1,000 nm. 
     
     
         52 . The assembly of  claim 46  wherein said first membrane-associated protein component comprises a signal protein or a component of a signal protein. 
     
     
         53 . The assembly of  claim 52  wherein said signal protein is a phosphotransferase. 
     
     
         54 . The assembly of  claim 46  wherein said first membrane-associated protein component is a first transmembrane receptor component. 
     
     
         55 . The assembly of  claim 54  wherein said first membrane-associated protein component is a first cytoplasmic domain of a transmembrane receptor. 
     
     
         56 . The assembly of  claim 46  wherein said assembly further comprises a plurality of a second membrane-associated protein component coupled to the membrane. 
     
     
         57 . The assembly of  claim 56  wherein said first membrane-associated protein component is a first transmembrane receptor component and said second membrane-associated protein component is a second transmembrane receptor component. 
     
     
         58 . The assembly of  claim 57  wherein said first membrane-associated protein component is a first cytoplasmic domain of a transmembrane receptor. 
     
     
         59 . The assembly of  claim 58  wherein said second membrane-associated protein component is a second cytoplasmic domain of a transmembrane receptor. 
     
     
         60 . A functional receptor complex, comprising: a lipid membrane including a first lipid component; a plurality of a first receptor protein component; each said first receptor protein component comprising at least one amino acid residue with binding affinity for said first lipid component; and at least one molecule complexed with said first receptor protein component; wherein said receptor complex exhibits a greater biological function than said first receptor protein component and said at least one molecule in an aqueous medium in the absence of the lipid membrane. 
     
     
         61 . The complex of  claim 60  wherein said molecule comprises a member selected from the group consisting of a second protein component with binding affinity for said lipid membrane, a second protein component that interacts with said first receptor protein component, an adaptor protein that interacts with said first receptor protein component and combinations thereof. 
     
     
         62 . The complex of  claim 60  wherein said molecule comprises a member selected from the group consisting of a second protein component with binding affinity for said lipid membrane and a second protein component that interacts with said first receptor protein component. 
     
     
         63 . The complex of  claim 62  wherein one of said first receptor protein component and said second protein component operates as a substrate of another one of said first receptor protein component and said second protein component. 
     
     
         64 . The complex of  claim 62  wherein said complex further comprises an adaptor protein. 
     
     
         65 . The complex of  claim 62  wherein said complex further comprises one or more additional protein components that operates as a substrate of one of said first receptor protein component and said second protein component. 
     
     
         66 . The complex of  claim 60  wherein said membrane is a unilamellar vesicle. 
     
     
         67 . An in vitro assembly for enhancing the function of a membrane-associated receptor protein component, said assembly comprising: a lipid membrane comprising a first lipid component; a plurality of a first membrane-associated receptor protein component, said first membrane-associated receptor protein component having a binding affinity for said first lipid component and being coupled with said first lipid component; wherein said coupling enhances receptor function of the first membrane-associated receptor protein component compared to receptor function exhibited by the first membrane-associated receptor protein component in an aqueous medium in the absence of the lipid membrane.