Polypeptides that bind membrane proteins

Abstract

Polypeptides which bind to the helical transmembrane region of membrane proteins are disclosed, as are methods for the design of polypeptides that bind to the transmembrane region of membrane proteins. Also provided are methods for the use of the disclosed polypeptides in various applications, as well as products made through the practice of the instant methods.

What is claimed: 
     
         1 . A polypeptide that binds to a transmembrane protein;
 the polypeptide being substantially complementary to a template polypeptide;   the template polypeptide having a backbone, the sequence of which backbone is at least, in major proportion, the sequence of a portion of the helical transmembrane region of the transmembrane protein;   at least some of the amino acid side chains of the template polypeptide being threaded with different side chains in order to improve the similarity of the template polypeptide to the portion of the helical region of the transmembrane protein.   
     
     
         2 . The polypeptide of  claim 1  wherein the backbone is derived from a table of helical portions of transmembrane proteins. 
     
     
         3 . The polypeptide of  claim 1  wherein the sequence of the polypeptide that binds to said transmembrane protein is derived from the template polypeptide through employment of a repacking algorithm. 
     
     
         4 . The polypeptide of  claim 1  wherein the different side chains are selected to improve helix-helix interaction. 
     
     
         5 . The polypeptide of  claim 1  further comprising at least one water solubility enhancing function. 
     
     
         6 . The polypeptide of  claim 5  wherein the function is bonded to at least one of the carbon and nitrogen termini of the polypeptide. 
     
     
         7 . The polypeptide according to  claim 1  having the amino acid sequence AYV MLL PFF IGL LLG LIF GGA FWG PAR HL (SEQ ID NO:2). 
     
     
         8 . The polypeptide according to  claim 1  having the amino acid sequence XXX XIX XSF XXG TXX GXX XMF XX (SEQ ID NO:7). 
     
     
         9 . The polypeptide according to  claim 8  having the amino acid sequence AYV FIL LSF ILG TLL GFL VMF WA (SEQ ID NO:8). 
     
     
         10 . The polypeptide according to  claim 1  comprising at least one d-amino acid. 
     
     
         11 . The polypeptide according to  claim 1  comprising a d-peptide. 
     
     
         12 . The polypeptide according to  claim 1  comprising a d-peptide having the amino acid sequence XXWFXXFXXIFXGFXXGXXTXXXQX (SEQ ID NO:10). 
     
     
         13 . The polypeptide according to  claim 12  having the amino acid sequence RAWFALFLLIFLGFLLGVATLLVQY (SEQ ID NO:11). 
     
     
         14 . A pharmaceutical composition comprising the polypeptide of  claim 1  and a pharmaceutically acceptable carrier, diluent, or excipient. 
     
     
         15 . A method for analyzing the ability of a substance to affect a function of a membrane protein having a helical transmembrane region comprising:
 adhering a template polypeptide to a surface under conditions selected to maintain the function of said template polypeptide;
 the template polypeptide having a backbone, the sequence of which backbone is at least, in major proportion, the sequence of a portion of the helical transmembrane region of the membrane protein; 
 at least some of the amino acid side chains of the template polypeptide being threaded with different side chains in order to improve the similarity of the template polypeptide to the portion of the helical region of the membrane protein; 
   contacting said substance with said template polypeptide or a part thereof; and,   measuring the function of said template polypeptide.   
     
     
         16 . A method for localizing a target protein in a sample, the target protein being a membrane protein having a helical transmembrane region, comprising:
 contacting a polypeptide with said sample,
 the polypeptide being substantially complementary to a template polypeptide; 
 the template polypeptide having a backbone, the sequence of which backbone is at least, in major proportion, the sequence of a portion of the helical transmembrane region of the membrane protein; 
 at least some of the amino acid side chains of the template polypeptide being threaded with different side chains in order to improve the similarity of the template polypeptide to the portion of the helical region of the membrane protein and, 
   detecting the presence of said polypeptide in said sample.   
     
     
         17 . The method according to  claim 16  wherein said polypeptide is conjugated to a detection label. 
     
     
         18 . A method for the quantitation of a target protein in a sample, the target protein being a membrane protein having a helical transmembrane region, comprising:
 contacting said sample with a polypeptide,
 the polypeptide being substantially complementary to a template polypeptide; 
 the template polypeptide having a backbone, the sequence of which backbone is at least, in major proportion, the sequence of a portion of the helical transmembrane region of the membrane protein; 
 at least some of the amino acid side chains of the template polypeptide being threaded with different side chains in order to improve the similarity of the template polypeptide to the portion of the helical region of the membrane protein; and, 
   measuring the presence of said polypeptide in said sample.   
     
     
         19 . The method according to  claim 18  wherein said polypeptide is conjugated to a detection label. 
     
     
         20 . The method according to  claim 18 , wherein the polypeptide is bound to a solid surface within a contacting environment, the sample is introduced into contacting environment, and an antibody specific to said target protein is introduced into the contacting environment, wherein said antibody is conjugated to a detection label. 
     
     
         21 . A method for analyzing the function of a membrane protein having a helical transmembrane region comprising:
 contacting said membrane protein with a polypeptide;
 the polypeptide being substantially complementary to a template polypeptide; 
 the template polypeptide having a backbone, the sequence of which backbone is at least, in major proportion, the sequence of a portion of the helical transmembrane region of the membrane protein; 
 at least some of the amino acid side chains of the template polypeptide being threaded with different side chains in order to improve the similarity of the template polypeptide to the portion of the helical region of the membrane protein; 
   determining whether any modulation of said membrane protein results subsequent to said contacting step.   
     
     
         22 . A method of protein purification comprising:
 passing a composition containing a target protein through a chromatography column;
 the target protein being a membrane protein having a helical transmembrane region; 
   said column containing an immobilized polypeptide;
 the polypeptide being substantially complementary to a template polypeptide; 
 the template polypeptide having a backbone, the sequence of which backbone is at least, in major proportion, the sequence of a portion of the helical transmembrane region of the membrane protein; and, 
 at least some of the amino acid side chains of the template polypeptide being threaded with different side chains in order to improve the similarity of the template polypeptide to the portion of the helical region of the membrane protein. 
   
     
     
         23 . A method of protein detection comprising:
 transferring a composition containing a fractionationed target protein to a solid support membrane, the target protein being a membrane protein having a helical transmembrane region;   said composition having been subjected to conditions suitable to cause denaturation of any proteins contained therein and to electrophoresis;   contacting said solid support membrane with a solution containing a polypeptide;
 the polypeptide being substantially complementary to a template polypeptide; 
 the template polypeptide having a backbone, the sequence of which backbone is at least, in major proportion, the sequence of a portion of the helical transmembrane region of the membrane protein; and, 
 at least some of the amino acid side chains of the template polypeptide being threaded with different side chains in order to improve the similarity of the template polypeptide to the portion of the helical region of the membrane protein. 
   
     
     
         24 . The method according to  claim 23  wherein said polypeptide is conjugated to a detection label. 
     
     
         25 . A method for determining the expression of a target protein in a cell sample, the target protein being a membrane protein having a helical transmembrane region, comprising:
 contacting said cell sample with a polypeptide;
 the polypeptide being substantially complementary to a template polypeptide; 
 the template polypeptide having a backbone, the sequence of which backbone is at least, in major proportion, the sequence of a portion of the helical transmembrane region of the membrane protein; and, 
 at least some of the amino acid side chains of the template polypeptide being threaded with different side chains in order to improve the similarity of the template polypeptide to the portion of the helical region of the membrane protein; and, 
   using flow cytometry to obtain an absorbance or fluorescence profile of said cell sample, wherein said absorbance or fluorescence profile varies depending on the absence or presence of a detection label in association with said polypeptide.   
     
     
         26 . The method according to  claim 25  wherein said detection label is conjugated to said polypeptide. 
     
     
         27 . A method for screening an expression library in order to isolate candidate genes that express a target protein, the target protein being a membrane protein having a helical transmembrane region, comprising:
 contacting a polypeptide with the protein expressed by a candidate gene of said expression library;
 the polypeptide being substantially complementary to a template polypeptide; 
 the template polypeptide having a backbone, the sequence of which backbone is at least, in major proportion, the sequence of a portion of the helical transmembrane region of the membrane protein; 
 at least some of the amino acid side chains of the template polypeptide being threaded with different side chains in order to improve the similarity of the template polypeptide to the portion of the helical region of the membrane protein; and, 
   assessing the affinity of said polypeptide for said protein expressed by said candidate gene.   
     
     
         28 . A method of delivering a drug to a cell expressing a target protein, the target protein being a membrane protein having a helical transmembrane region, comprising:
 contacting said cell with a polypeptide,
 the polypeptide being conjugated to a drug, and being substantially complementary to a template polypeptide; 
 the template polypeptide having a backbone, the sequence of which backbone is at least, in major proportion, the sequence of a portion of the helical transmembrane region of the membrane protein; and, 
 at least some of the amino acid side chains of the template polypeptide being threaded with different side chains in order to improve the similarity of the template polypeptide to the portion of the helical region of the membrane protein. 
   
     
     
         29 . A method of measuring association between a polypeptide and a helical transmembrane region of a membrane protein,
 the polypeptide being substantially complementary to a template polypeptide;   the template polypeptide having a backbone, the sequence of which backbone is at least, in major proportion, the sequence of a portion of the helical transmembrane region of the membrane protein;   at least some of the amino acid side chains of the template polypeptide being threaded with different side chains in order to improve the similarity of the template polypeptide to the portion of the helical region of the membrane protein;   
       comprising:
 titrating said polypeptide with said membrane protein or a portion thereof, wherein said polypeptide is labeled with one of a donor fluorophore and an acceptor fluorophore and said membrane protein or a portion thereof is labeled with the other of a donor fluorophore and an acceptor fluorophore, and, 
 measuring the degree of quenching of fluorescent emission from said donor fluorophore or the degree of increase of fluorescent emission from said acceptor fluorophore, or both. 
 
     
     
         30 . A method of assessing the ability of a polypeptide to insert into a phospholipid bilayer membrane,
 the polypeptide being substantially complementary to a template polypeptide;   the template polypeptide having a backbone, the sequence of which backbone is at least, in major proportion, the sequence of a portion of the helical transmembrane region of the membrane protein;   at least some of the amino acid side chains of the template polypeptide being threaded with different side chains in order to improve the similarity of the template polypeptide to the portion of the helical region of the membrane protein;   
       comprising:
 measuring the emission maximum of tryptophan (Trp) residues in said polypeptide in order to obtain a first emission value; 
 contacting said polypeptide with structures having a hydrophobic region; 
 after said contacting, measuring the emission maximum of tryptophan (Trp) residues in said polypeptide to obtain a second emission value; and, 
 comparing said first emission value with said second emission value. 
 
     
     
         31 . A method of assessing the potential for a polypeptide to cause cell lysis,
 the polypeptide being substantially complementary to a template polypeptide;   the template polypeptide having a backbone, the sequence of which backbone is at least, in major proportion, the sequence of a portion of the helical transmembrane region of the membrane protein;   at least some of the amino acid side chains of the template polypeptide being threaded with different side chains in order to improve the similarity of the template polypeptide to the portion of the helical region of the membrane protein;   
       comprising:
 contacting said polypeptide with a cell sample to produce a test sample; and, 
 determining the extent of cell lysis in said test sample. 
 
     
     
         32 . The method according to  claim 31  wherein said determining comprises centrifuging said test sample to produce a supernatant, followed by measuring the optical density (OD) of said supernatant. 
     
     
         33 . A method of modulating the function of a transmembrane protein comprising contacting a membrane containing said transmembrane protein with a polypeptide;
 the polypeptide being substantially complementary to a template polypeptide;   the template polypeptide having a backbone, the sequence of which backbone is at least, in major proportion, the sequence of a portion of the helical transmembrane region of the transmembrane protein;   at least some of the amino acid side chains of the template polypeptide being threaded with different side chains in order to improve the similarity of the template polypeptide to the portion of the helical region of the transmembrane protein.