# US 8,541,190 B2 · Methods and materials for in vitro analysis and/or use of membrane-associated proteins, portions thereof or variants thereof > Agents: this data is available as JSON from the patentgrep API and MCP server — see https://patentgrep.com/llms.txt - Number: US 8,541,190 B2 - Document ID: US8541190B2 - Status: Granted - Kind: B2 - Application number: 11/823,728 - Filed: 2007-06-28 - Published: 2008-01-03 - Granted: 2013-09-24 - Earliest claimed: 2006-06-28 - Applicants: Robert M. Weis; Anthony L. Shrout; Edward A. Esposito - Inventors: Robert M. Weis; Anthony L. Shrout; Edward A. Esposito - Web page: https://patentgrep.com/patent/US8541190B2 ## CPC - G01N 33/544: the carrier being organic - G01N: INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES (measuring or testing apparatus or processes other than immunoassay, involving enzymes or microorganisms C12M, C12Q) ## Abstract Methods and materials use template-directed assembly of polypeptides and optionally additional reagents to analyze the functionality of membrane-associated proteins, such as, for example, portions of transmembrane proteins, membrane-associated proteins, and others proteins that bind to transmembrane proteins and membrane-associated proteins, and to analyze the effect of test compounds or mutations on the functionality of same. The methods and materials of the present application provide a more native-like environment for analyzing the functionality of membrane-associated proteins, and thus provide effective tools for studies involving the detection of the level of enzyme activity of such proteins in an environment that closely resembles the native environment in the cell, and for novel manufacturing processes. ## Claims ### Claim 1 A method for analyzing in vitro the effect of a molecule upon a polypeptide-catalyzed reaction or cascade, comprising: providing an aqueous fluid including: one or more reagent; and a biologically active complex including a synthetic lipid membrane-like template and at least one membrane-associated polypeptide attached to the template, wherein the complex is functional under a given set of conditions to produce a measurable modification in the content of said one or more reagent or in said polypeptide; introducing a fluid a test molecule selected from the group consisting of a drug, a drug candidate, an agonist and an antagonist; and measuring the modification to determine the effect of the test molecule on the reaction or cascade. ### Claim 2 (depends on 1) The method in accordance with claim 1 wherein the test molecule is selected from the group consisting of a drug, a drug candidate. ### Claim 3 (depends on 1) The method in accordance with claim 1 wherein the measurable modification results from a process selected from the group consisting of (1) a chemical modification to the polypeptide resulting from intrinsic enzymatic activity of the polypeptide as it interacts with the template, (2) chemical modification of a soluble substrate reagent present in the fluid that is catalyzed by the polypeptide as it interacts with the template, (3) chemical modification of a soluble substrate reagent that is catalyzed by enzymatic activity of a signaling enzyme present in the fluid that is recruited to the complex, (4) chemical modification to the polypeptide in a process catalyzed by a signaling protein that is recruited to the complex, and (5) chemical modification of a soluble substrate reagent present in the fluid that results from a reaction cascade initiated by the polypeptide as it interacts with the template or a signaling enzyme that is recruited to the complex. ### Claim 4 (depends on 3) The method in accordance with claim 3 wherein the polypeptide comprises a receptor tyrosine kinase domain, and wherein the process comprises autophosphorylation of the receptor tyrosine kinase domain. ### Claim 5 (depends on 1) The method in accordance with claim 1 wherein the measurable modification is a modification selected from the group consisting of phosphorylation, dephosphorylation, acetylation, methylation, acylation, glycosylation, glycosylphosphatidylinositol (GPI) anchoring, sulfation, disulfide bond formation, deamidation, ubiquitination, sumoylation nitration of tyrosine, hydrolysis of ATP or GTP activation of a fluorescent signal, release of a reaction product and utilization of a reagent initially present in the fluid. ### Claim 6 (depends on 1) The method in accordance with claim 1 wherein the template is supported on a solid substrate material. ### Claim 7 (depends on 6) The method in accordance with claim 6 wherein the substrate is selected from the group consisting of a glass slide, a glass bead, a silicon wafer, a silicon chip, a planar noble metal, a colloidal noble metal, a metal oxide layer, a nanoparticulate material, a polymer slab, a polymer film and a polymer bead. ### Claim 8 (depends on 6) The method in accordance with claim 6 wherein the template is selected from the group consisting of a phospholipid bilayer, a phospholipid monolayer and a polymer film. ### Claim 9 (depends on 1) The method in accordance with claim 1 wherein the polypeptide has attached thereto a linker component effective to attach the polypeptide to the template. ### Claim 10 (depends on 9) The method in accordance with claim 9 wherein the linker component is selected from the group consisting of a component effective to covalently bond to the template, a component effective to interact with the template noncovalently by metal chelation, a component effective to interact with the template noncovalently by other complementary interactions, and an insertion domain effective to interact with the template noncovalently by insertion of at least a portion of the domain into the template. ### Claim 11 (depends on 9) The method in accordance with claim 9 wherein the linker component comprises a component effective to interact with the template noncovalently by metal chelation, and wherein the metal or metal ion is associated with the template. ### Claim 12 (depends on 9) The method in accordance with claim 9 wherein the linker component comprises a component effective to interact with the template noncovalently by metal chelation, and wherein the metal or metal ion is associated with the linker component. ### Claim 13 (depends on 9) The method in accordance with claim 9 wherein the linker component comprises a genetically engineered histidine tag. ### Claim 14 (depends on 9) The method in accordance with claim 9 wherein the linker component comprises an insertion domain. ### Claim 15 (depends on 14) The method in accordance with claim 14 wherein the insertion domain is effective to interact with the template noncovalently by insertion of at least a portion of the domain into the template, and wherein at least a portion of the insertion domain interacts with the template by hydrophobic interactions. ### Claim 16 (depends on 14) The method in accordance with claim 14 wherein the insertion domain comprises a genetically engineered peptidyl insertion domain. ### Claim 17 (depends on 14) The method in accordance with claim 14 wherein the insertion domain comprises an anchoring moiety formed by the adaptation of naturally occurring mechanisms. ### Claim 18 (depends on 17) The method in accordance with claim 17 wherein the naturally occurring mechanism is selected from the group consisting of palmitoylation, myristoylation, prenylation, geranylation, GPI linkage and a synthetic analog thereof. ### Claim 19 A method for analyzing in vitro the effect of a molecule upon a polypeptide-catalyzed reaction or cascade, comprising: providing an aqueous fluid including: one or more reagent; and a biologically active complex including a synthetic lipid membrane-like template and at least one membrane-associated polypeptide attached to the template, wherein the complex is functional under a given set of conditions to produce a measureable modification in the content of said one or more reagent or in said polypeptide; introducing into the fluid a test molecule selected from the group consisting of a drug, a drug candidate, an agonist and an antagonist; and measuring the modification to determine the effect of the test molecule on the reaction or cascade; wherein the template is a free-standing template. ### Claim 20 (depends on 19) The method in accordance with claim 19 wherein the template is selected from the group consisting of a lipid vesicle, a polymer vesicle, a polymer micelle, a polymer molecule, and a polymer bead.