Methods for dye selection for protein melt temperature determinations

Abstract

According to the present teachings, compositions, kits, and methods for protein melt analysis are provided that utilizing one or more fluorophore dyes. In some embodiments, a method comprises preparing a sample by mixing at least one protein with two or more dyes, and applying a controlled heating, while recording the fluorescence emission of the sample. The methods can be used, for example, for screening conditions for optimized protein stability, screening for ligands that bind and enhance protein stability (e.g., protein-protein interactions), screening for mutations for enhanced stability, screening crystallization conditions for protein stability, screening storage conditions for protein stability, and screening conditions in which a protein will be used (e.g., production conditions, treatment conditions, etc.) for protein stability.

1 .- 18 . (canceled) 
     
     
         19 . A composition comprising at least one protein, and a mixture of at least two fluorophore dyes, wherein each of the at least two fluorophore dyes is configured to provide at least a minimally fluorescent signal when the at least one protein is present in its native state and when the at least one protein is present in an unfolded state, then each of the at least two fluorophore dyes is configured to provide a substantially increased fluorescent signal. 
     
     
         20 . The composition of  claim 19 , wherein one of the at least two fluorophore dyes is Nile Red. 
     
     
         21 . The composition of  claim 19 , wherein one of the at least two fluorophore dyes is a styryl dye. 
     
     
         22 . The composition of  claim 21 , wherein the styryl dye has a structure of the formula:
   Q a -B-M a ;  a.)
   wherein Q a  has a structure of the formula:   
       
         
           
           
               
               
           
         
         X is —S—, —O—, —NR 17 —, or —CR 7 R 8 ; 
         R 7  and R 8  are optionally and independently H, Cl, F, phenyl, or C 1 -C 6  alkyl; or R 7  and R 8  taken in combination form a 5- or 6-membered saturated ring; 
         R 1  and R 2  are optionally and independently H, Cl, F, sulfo, carboxy, salt of sulfo, salt of carboxy, C 1 -C 6  alkyl or C 1 -C 6  alkoxy; or R 1  and R 2 , when taken in combination, form a fused 6-membered aromatic ring that is optionally further substituted one or more times by H, Cl, F, C 1 -C 6  alkyl or C 1 -C 6  alkoxy, carboxy, sulfo or salts of carboxy or sulfo; 
         R 3 , R 4 , R 5 , and R 6  are optionally and independently H, Cl, F, C 1 -C 6  alkyl or C 1 -C 6  alkoxy; or any two adjacent R 3 , R 4 , R 5  and R 6 , when taken in combination, form a fused 6-membered aromatic ring that is optionally further substituted one or more times by H, Cl, F, C 1 -C 6  alkyl or C 1 -C 6  alkoxy, carboxy, sulfo or salts of carboxy or sulfo; 
         TAIL is attached to the nitrogen atom of Q a  through a carbon atom and contains a total of 1-22 non-hydrogen atoms, wherein the non-hydrogen atoms are selected from the group consisting of C, O, N or S, such that within TAIL each heteroatom is separated from any adjacent heteroatoms by at least two carbon atoms, and further wherein TAIL is composed of bonds selected from the group consisting of carbon-carbon bonds (C—C), ether bonds (C—O—C), thioether bonds (C—S—C) or amine bonds (C—NR 9 —C); 
         wherein any carbon atom in TAIL is optionally further substituted by hydroxy, carboxy, sulfo, amino, or ammonium, and wherein any amine bond, amino or ammonium in TAIL is optionally substituted by an R 9  that is C 2 -C 6  alkyl optionally further substituted by hydroxy, carboxy, sulfo, amino, amino substituted by 1-2 C 1 -C 6  alkyls or ammonium substituted by 1-3 C 1 -C 6  alkyls; 
         or the nitrogen atoms of TAIL form either one or two saturated 5- or 6-membered rings in combination with other C or N atoms in TAIL; 
         B has a structure of the formula:
   —(CR 7 ═CR 8 ) n —;
 
 
         wherein n is an integer of 1-3; 
         M a  has a structure of the formula: 
       
       
         
           
           
               
               
           
         
         R 11  and R 12  are independently H, F, Cl, or —CH 3 , 
         the ring substituents not specified are hydrogen; 
         R 13  and R 14  are independently C 1 -C 8  alkyls that are linear, branched, saturated or unsaturated, and are optionally substituted one or more times by F, hydroxy or C 1 -C 6  alkoxy; or R 13  and R 14 , when taken in combination, form a 5- or 6-membered saturated ring that contains 0 or 1 oxygen heteroatom; or R 11  taken in combination with R 13  and R 10  taken in combination with R 12  are independently —(CH 2 ) 2 — or —(CH 2 ) 3 —; or
   Q b -B-M b ;  b.)
 
 
         wherein Q b  has a structure of one of the following formulae: 
       
       
         
           
           
               
               
           
         
         wherein X is —S—, —O—, —NR 17 —, or —CR 7 R 8 ; 
         R 7  and R 8  are independently H, Cl, F, phenyl, or C 1 -C 6  alkyl; or R 7  and R 8  taken in combination form a 5- or 6-membered saturated ring; 
         R 1  and R 2  are independently H, Cl, F, sulfo, carboxy, salt of sulfo, salt of carboxy, C 1 -C 6  alkyl or C 1 -C 6  alkoxy; or R 1  and R 2 , when taken in combination, form a fused 6-membered aromatic ring optionally further substituted one or more times by H, Cl, F, C 1 -C 6  alkyl or C 1 -C 6  alkoxy, carboxy, sulfo or salts of carboxy or sulfo; 
         R 3 , R 4 , R 5 , and R 6  are independently H, Cl, F, C 1 -C 6  alkyl or C 1 -C 6  alkoxy; or any two adjacent substituents of R 3 , R 4 , R 5  and R 6 , when taken in combination, form a fused 6-membered aromatic ring optionally further substituted one or more times by H, Cl, F, C 1 -C 6  alkyl or C 1 -C 6  alkoxy, carboxy, sulfo or salts of carboxy or sulfo; 
         TAIL is attached to the nitrogen atom of Q b  through a carbon atom and is —(CH 2 ) m SO 3   −  or —(CH 2 ) m C0 2   − , m=1 to 6, and the negative charge is balanced by a positive charge on the nitrogen heterocycle Q b ; or 
         TAIL is —(CH 2 ) m NR a R b  or —(CH 2 ) m N + R a R b R c , where R a , R b  and R c , which may be the same or different, are H or C 1 -C 6  alkyl or R a  and R b  taken in combination form a 3-6 membered ring, optionally containing a heteroatom that is O, NR d  or S, where R d  is H or C 1 -C 6  alkyl; 
         B has the formula —(CH═CH) n —, wherein n has a value of 1-3; 
         M b  has a structure of the formula: 
       
       
         
           
           
               
               
           
         
         wherein R 11  is C 1 -C 6  alkyl, phenyl, or phenyl substituted one or more times by H, Cl, F, C 1 -C 6  alkyl or C 1 -C 6 -alkoxy; and 
         R 12 , R 13 , R 14  and R 15  are H, Cl, F, C 1 -C 6 alkyl or C 1 -C 6  alkoxy. 
       
     
     
         23 . The composition of  claim 19 , wherein the at least one protein comprises a protein/ligand complex. 
     
     
         24 . The composition of  claim 23 , wherein the protein/ligand complex is an antibody/antigen complex. 
     
     
         25 . The composition of  claim 23 , wherein the ligand is a peptide, a polynucleotide, or an aptamer 
     
     
         26 . A kit comprising a composition comprising at least two fluorophore dyes, wherein each of the at least two fluorophore dyes is configured to provide at least a minimally fluorescent signal when at least one protein is in its native state and when the at least one protein is present in an unfolded state, then each of the at least two fluorophore dyes is configured to provide a substantially increased fluorescent signal. 
     
     
         27 . The kit of  claim 26 , wherein one of the at least two fluorophore dyes is Nile Red. 
     
     
         28 . The kit of  claim 26 , wherein one of the at least two fluorophore dyes is a styryl dye. 
     
     
         29 . The kit of  claim 28 , wherein the styryl dye has a structure of the formula
   Q a -B-M a ;  A.)
   wherein Q a  has a structure of the formula:   
       
         
           
           
               
               
           
         
         X is —S—, —O—, —NR 17 —, or —CR 7 R 8 ; 
         R 7  and R 8  are optionally and independently H, Cl, F, phenyl, or C 1 -C 6  alkyl; or R 7  and R 8  taken in combination form a 5- or 6-membered saturated ring; 
         R 1  and R 2  are optionally and independently H, Cl, F, sulfo, carboxy, salt of sulfo, salt of carboxy, C 1 -C 6  alkyl or C 1 -C 6  alkoxy; or R 1  and R 2 , when taken in combination, form a fused 6-membered aromatic ring that is optionally further substituted one or more times by H, Cl, F, C 1 -C 6  alkyl or C 1 -C 6  alkoxy, carboxy, sulfo or salts of carboxy or sulfo; 
         R 3 , R 4 , R 5 , and R 6  are optionally and independently H, Cl, F, C 1 -C 6  alkyl or C 1 -C 6  alkoxy; or any two adjacent R 3 , R 4 , R 5  and R 6 , when taken in combination, form a fused 6-membered aromatic ring that is optionally further substituted one or more times by H, Cl, F, C 1 -C 6  alkyl or C 1 -C 6  alkoxy, carboxy, sulfo or salts of carboxy or sulfo; 
         TAIL is attached to the nitrogen atom of Q a  through a carbon atom and contains a total of 1-22 non-hydrogen atoms, wherein the non-hydrogen atoms are selected from the group consisting of C, O, N or S, such that within TAIL each heteroatom is separated from any adjacent heteroatoms by at least two carbon atoms, and further wherein TAIL is composed of bonds selected from the group consisting of carbon-carbon bonds (C—C), ether bonds (C—O—C), thioether bonds (C—S—C) or amine bonds (C—NR 9 —C); 
         wherein any carbon atom in TAIL is optionally further substituted by hydroxy, carboxy, sulfo, amino, or ammonium, and wherein any amine bond, amino or ammonium in TAIL is optionally substituted by an R 9  that is C 2 -C 6  alkyl optionally further substituted by hydroxy, carboxy, sulfo, amino, amino substituted by 1-2 C 1 -C 6  alkyls or ammonium substituted by 1-3 C 1 -C 6  alkyls; 
         or the nitrogen atoms of TAIL form either one or two saturated 5- or 6-membered rings in combination with other C or N atoms in TAIL; 
         B has a structure of the formula:
   —(CR 7 ═CR 8 ) n —;
 
 
         wherein n is an integer of 1-3, 
         M a  has a structure of the formula: 
       
       
         
           
           
               
               
           
         
         R 11  and R 12  are independently H, F, Cl, or —CH 3 , R 13  and R 14  are independently C 1 -C 8  alkyls that are linear, branched, saturated or unsaturated, and are optionally substituted one or more times by F, hydroxy or C 1 -C 6  alkoxy; or R 13  and R 14 , when taken in combination, form a 5- or 6-membered saturated ring that contains 0 or 1 oxygen heteroatom; or R 11  taken in combination with R 13  and R 10  taken in combination with R 12  is independently —(CH 2 ) 2 — or —(CH 2 ) 3 —; or
   Q b -B-M b ;  B.)
 
 
         wherein Q b  has a structure of one of the following formulae: 
       
       
         
           
           
               
               
           
         
         wherein X is —S—, —O—, —NR 17 —, or —CR 7 R 8 ; 
         R 7  and R 8  are independently H, Cl, F, phenyl, or C 1 -C 6  alkyl; or R 7  and R 8  taken in combination form a 5- or 6-membered saturated ring; 
         R 1  and R 2  are independently H, Cl, F, sulfo, carboxy, salt of sulfo, salt of carboxy, C 1 -C 6  alkyl or C 1 -C 6  alkoxy; or R 1  and R 2 , when taken in combination, form a fused 6-membered aromatic ring optionally further substituted one or more times by H, Cl, F, C 1 -C 6  alkyl or C 1 -C 6  alkoxy, carboxy, sulfo or salts of carboxy or sulfo; 
         R 3 , R 4 , R 5 , and R 6  are independently H, Cl, F, C 1 -C 6  alkyl or C 1 -C 6  alkoxy; or any two adjacent substituents of R 3 , R 4 , R 5  and R 6 , when taken in combination, form a fused 6-membered aromatic ring optionally further substituted one or more times by H, Cl, F, C 1 -C 6  alkyl or C 1 -C 6  alkoxy, carboxy, sulfo or salts of carboxy or sulfo; 
         TAIL is attached to the nitrogen atom of Q b  through a carbon atom and is —(CH 2 ) m SO 3   −  or —(CH 2 ) m C0 2   − , m=1 to 6, and the negative charge is balanced by a positive charge on the nitrogen heterocycle Q b ; or 
         TAIL is —(CH 2 ) m NR a R b  or —(CH 2 ) m N + R a R b R c , where R a , R b  and R c , which may be the same or different, are H or C 1 -C 6  alkyl or R a  and R b  taken in combination form a 3-6 membered ring, optionally containing a heteroatom that is O, NR d  or S, where R d  is H or C 1 -C 6  alkyl; 
         B has the formula —(CH═CH) n —, wherein n has a value of 1-3; 
         M b  has a structure of the formula: 
       
       
         
           
           
               
               
           
         
         wherein R 11  is C 1 -C 6  alkyl, phenyl, or phenyl substituted one or more times by H, Cl, F, C 1 -C 6  alkyl or C 1 -C 6 -alkoxy; and 
         R 12 , R 13 , R 14  and R 15  are H, 1I, F, C 1 -C 6 alkyl or C 1 -C 6  alkoxy.