Leucine beta roll domains and uses thereof

Abstract

In one aspect, the invention relates to a peptide that forms a calcium-dependent hydrogel using a rationally engineered beta roll peptide. In the absence of calcium, the peptide is intrinsically disordered. Upon addition of calcium, the peptide forms a corkscrew-like structure. In one embodiment, one face of the beta roll is mutated to comprise leucine residues. In some embodiments, a leucine zipper forming helical domain to the engineered beta roll forms hydrogels by physical cross-linking in calcium rich environments.

1 . A beta roll domain comprising at least one repeating unit from the wild-type RTX domain of adenylate cyclase from  Bordetella pertussis  that has been modified to include at least one modification, said modified repeat unit including an amino acid sequence X 1 X 2 X 3 X 4 X 5 X 6 X 7 X 8 X 9  adhering to the following sequence definition:
 a) X 1  is an amino acid selected from the group consisting of glycine, valine and serine;   b) X 2  is an amino acid selected from the group consisting of glycine, serine, aspartic acid and leucine;   c) X 3  is an amino acid selected from the group consisting of alanine, glutamic acid, glutamine, tyrosine and glycine;   d) X 4  is an amino acid selected from the group consisting of glycine and arginine;   e) X 5  is an amino acid selected from the group consisting of aspartic acid, alanine, asparagine, serine, and histidine;   f) X 6  is an amino acid selected from the group consisting of aspartic acid and asparagine; and   g) wherein at least one of X 7 , X 8 , and X 9  is mutated to a leucine residue from a non-leucine residue as found in a corresponding X 7 , X 8 , and X 9  position of the at least one repeating unit from the wild-type RTX domain of adenylate cyclase from  Bordetella pertussis.      
     
     
         2 . The beta roll of  claim 1 , wherein the beta roll domain further comprises X 10 , wherein X 10  is appended adjacent to X 9 , and wherein
 (a) X 10  is the amino acid glutamic acid.   
     
     
         3 . The beta roll of  claim 1 , wherein the beta roll domain further comprises X 10 X 11 , wherein X 10  is appended adjacent to X 9 , and
 a) X 10  is the amino acid phenylalanine; and   b) X 11  is the amino acid glycine.   
     
     
         4 . The beta roll of  claim 1 , wherein X 1  is the amino acid glycine. 
     
     
         5 . The beta roll of  claim 1 , wherein X 4  is the amino acid glycine. 
     
     
         6 . The beta roll of  claim 1 , wherein X 6  is the amino acid aspartic acid. 
     
     
         7 . (canceled) 
     
     
         8 . The beta roll of  claim 4 , wherein X 4  is the amino acid glycine. 
     
     
         9 . The beta roll of  claim 4 , wherein X 6  is the amino acid aspartic acid. 
     
     
         10 . The beta roll of  claim 4 , wherein X 8  is the amino acid leucine. 
     
     
         11 . The beta roll of  claim 5 , wherein X 6  is the amino acid aspartic acid. 
     
     
         12 . The beta roll of  claim 1 , wherein
 a) X 7  is the amino acid leucine;   b) X 8  is the amino acid leucine; and   c) X 9  is the amino acid leucine.   
     
     
         13 . The beta roll of  claim 1 , wherein the amino acid sequence is GSARDDVLI, GDAGANLLL, GLAGNDVLS, GGAGDDLLL, GDEGSDLLS, GDAGNDLLL, GGQGDDTYLFG, VGYGHDLILE, or SGGGHDTIR. 
     
     
         14 . The beta roll of  claim 1 , wherein the amino acid sequence is GDAGANLLL, GGAGDDLLL, GDAGNDLLL, or VGYGHDLILE. 
     
     
         15 . A protein hydrogel network comprising the beta roll of  claim 1 . 
     
     
         16 . The protein hydrogel network of  claim 15 , wherein the beta roll is fused to a leucine zipper with a soluble linker region. 
     
     
         17 . (canceled) 
     
     
         18 . (canceled) 
     
     
         19 . A beta roll comprising a modified scaffold from the wild-type RTX domain of adenylate cyclase from  Bordetella pertussis,  wherein non-hydrophobic amino acids were mutated to hydrophobic amino acids. 
     
     
         20 . The beta roll of  claim 19 , wherein the engineered hydrophobic amino acids project radially outward to create a hydrophobic face suitable for dimerization, in the presence of calcium. 
     
     
         21 . A protein hydrogel network comprising the beta roll of  claim 19 . 
     
     
         22 . The protein hydrogel network of  claim 21 , wherein the hydrogel network is formed only in calcium rich environments where the folded leucine beta roll domains provide the necessary cross-linking interface. 
     
     
         23 . The protein hydrogel network of  claim 21 , wherein the beta roll is fused to a leucine zipper with a soluble linker region.