Novel polypeptide scaffolds and use thereof

Abstract

Novel peptide having a sequence according to SEQ. ID. No. 1, SEQ. ID. No. 2 and/or SEQ. ID. No. 3 are disclosed and also polypeptide scaffold consisting of a four helix bundle formed of two dimerized helix-loop-helix motifs, said helix-loop-helix motifs having sequences according to SEQ. ID. No. 1, SEQ. ID. No. 2 and/or SEQ. ID. No. 3 which may comprise a fluorescent probe at the side chain of Lys15 and a ligand with affinity for a target molecule at the side chain of Lys8 or Lys34. Also disclosed are polypeptide scaffolds for use in biosensing applications with or without the polypeptide scaffold anchored to a solid surface.

1 .- 27 . (canceled) 
     
     
         28 . A method for detecting a target molecule or ion in a liquid sample by
 bringing the liquid sample in contact with a polypeptide comprising a four-helix bundle formed of two dimerized helix-loop-helix motifs, wherein at least one of the helix-loop-helix motifs is attached to a ligand with affinity for the target molecule or ion, and at least one of the helix-loop-helix motifs is attached to a reporter group which gives rise to a measurable signal upon binding of said ligand to said target molecule or ion; and   detecting the signal produced as a result of bringing the liquid sample in contact with the polypeptide.   
     
     
         29 . The method of  claim 28 , wherein the polypeptide is attached to a surface of a vesicle, a micelle or a membrane. 
     
     
         30 . The method of  claim 28 , wherein the polypeptide is present in a solution in the wells of a microtiter plate or in indentations on a solid surface. 
     
     
         31 . The method of  claim 28 , wherein the polypeptide is present in an array on a solid surface. 
     
     
         32 . The method of  claim 28 , wherein the target molecule is a biomolecule. 
     
     
         33 . The method of  claim 28 , wherein the ligand is selected from the group consisting of peptides with affinity for a protein, proteins with affinity for a protein, inhibitors of an enzyme, agonists of a receptor protein, antagonists of a receptor protein, parts of DNA, parts of RNA, parts of PNA, carbohydrates, haptens, toxins, metabolites, hormones, ion chelating agents, drugs, steroids, lipids and combinations of two or more of such ligands. 
     
     
         34 . The method of  claim 28 , wherein the reporter group is a fluorescent probe. 
     
     
         35 . The method of  claim 34 , wherein the fluorescent probe is selected from the group consisting of dansyl, fluorescein, and rhodamin. 
     
     
         36 . The method of  claim 28 , wherein at least one of the two the helix-loop-helix motifs has a sequence independently selected from SEQ. ID. No. 1, SEQ. ID. No. 2 and SEQ. ID. No. 3. 
     
     
         37 . The method of  claim 28 , wherein each one of the two the helix-loop-helix motifs has a sequence independently selected from SEQ. ID. No. 1, SEQ. ID. No. 2 and SEQ. ID. No. 3. 
     
     
         38 . The method of  claim 36 , wherein the side chain of Lys 15 of at least one helix-loop-helix motif is attached to a ligand or a reporter group. 
     
     
         39 . The method of  claim 28 , wherein the polypeptide comprises at least one helix-loop-helix motif having SEQ. ID. No. 2, wherein the ligand with high affinity for a target molecule or ion is attached to the side chain of Lys34 in the at least one helix-loop-helix motif having SEQ. ID. No. 2. 
     
     
         40 . The method of  claim 28 , wherein the polypeptide comprises at least one helix-loop-helix motif having SEQ. M. No. 3, wherein the ligand with high affinity for a target molecule or ion is attached to the side chain of Lys8 in the at least one helix-loop-helix motif having SEQ. ID. No. 3. 
     
     
         41 . The method of  claim 28 , for use in the determination of the concentration of the target molecule or ion in the sample. 
     
     
         42 . The method of  claim 41 , performed by use of an array of polypeptides, attached to ligands of different affinities for the target molecule or ion. 
     
     
         43 . The method of  claim 28 , for use in the determination of the affinity for the ligand of the target molecule or ion. 
     
     
         44 . A method for determining the concentration of a target molecule or ion in a liquid sample by
 bringing the liquid sample in contact with an array of polypeptides attached to a number of spots on a solid surface or located in a number of wells or indentations on a solid surface, each polypeptide in the array comprising a four-helix bundle formed of two dimerized helix-loop-helix motifs, wherein in each polypeptide at least one of the helix-loop-helix motifs is attached to a ligand with affinity for the target molecule or ion, and wherein in each polypeptide at least one of the helix-loop-helix motifs is attached to a reporter group which gives rise to a measurable signal upon binding of said ligand to said target molecule or ion, wherein the ligands in at least two spots, wells or indentations of the array differ in affinity for the target molecule or ion; and   measuring the signals produced in the wells, spots or indentations of the array.   
     
     
         45 . An array of polypeptides attached to a number of spots on a solid surface or located in a number of wells or indentations on a solid surface, each polypeptide in the array comprising a four-helix bundle formed of two dimerized helix-loop-helix motifs, wherein in each polypeptide at least one of the helix-loop-helix motifs is attached to a ligand with affinity for the target molecule or ion, and wherein in each polypeptide at least one of the helix-loop-helix motifs is attached to a reporter group which gives rise to a measurable signal upon binding of said ligand to said target molecule or ion. 
     
     
         46 . The array of  claim 45 , wherein the ligands in at least two spots, wells or or indentations of the array differ in affinity for the target molecule or ion. 
     
     
         47 . The array of  claim 45 , wherein in each polypeptide said reporter group is a fluorescent probe.