Self-Assembling Protein Nanostructures

Abstract

Synthetic nanostructures, proteins that are useful, for example, in making synthetic nanostructures, and methods for designing such synthetic nanostructures are disclosed herein.

1 . An isolated nanostructure, comprising
 (a) a plurality of first proteins that self-interact to form a first multimeric substructure comprising at least one axis of rotational symmetry;   (b) a plurality of second proteins that self-interact to form a second multimeric substructure comprising at least one axis of rotational symmetry;   wherein multiple copies of the first multimeric substructure and the second multimeric substructure interact with each other at one or more symmetrically repeated, non-natural, non-covalent protein-protein interfaces that orient the first multimeric substructure and the second multimeric substructure such that their symmetry axes are aligned with symmetry axes of the same kind in a designated mathematical symmetry group.   
     
     
         2 . The nanostructure of  claim 1 , in which the mathematical symmetry group is selected from the group consisting of tetrahedral point group symmetry, octahedral point group symmetry, and icosahedral point group symmetry. 
     
     
         3 . The nanostructure of  claim 1 , wherein the first multimeric substructure comprises a dimer, trimer, tetramer, or pentamer of the first protein, and wherein the second multimeric substructure comprises a dimer or trimer of the second protein. 
     
     
         4 . The nanostructure of  claim 1 , wherein the first proteins and the second proteins are between 30-250 amino acids in length. 
     
     
         5 . The nanostructure of  claim 1 , wherein each symmetrically repeated instance of the non-natural, non-covalent protein-protein interface between the first multimeric substructure and the second multimeric substructure buries between 1000-2000 A 2  of solvent-accessible surface area (SASA) on the first multimeric substructure and the second multimeric substructure. 
     
     
         6 . The nanostructure of  claim 1 , wherein each symmetrically repeated, non-natural, non-covalent protein-protein interface between the first multimeric substructure and the second multimeric substructure has a shape complementary value between 0.5-0.8. 
     
     
         7 . The nanostructure of  claim 1 , wherein at least 50% of the atomic contacts comprising each symmetrically repeated, non-natural, non-covalent protein-protein interface between the first multimeric substructure and the second multimeric substructure is formed from amino acid residues residing in elements of alpha helix and/or beta strand secondary structure. 
     
     
         8 . A nanostructure produced by a method comprising:
 generating a plurality of representations of a first protein building block using a computing device;   generating a plurality of representations of a second protein building block using the computing device, wherein the first protein building block differs from the second protein building block;   generating an arrangement of the plurality of representations of the first protein building block and the plurality of representations of the second protein building block according to symmetric operations of a designated mathematical symmetry group using the computing device;   computationally determining a docked configuration of the arrangement of the plurality of representations of the first protein building block and the plurality of representations of the second protein building block by generating at least one interface for each protein building block of the arrangement that is suitable for computational protein-protein interface design using the computing device;   computationally modifying amino acid sequences of the plurality of representations of the first protein building block and the plurality of representations of the second protein building block in the docked configuration to specify a plurality of representations of protein-protein interfaces, wherein the plurality of representations of protein-protein interfaces comprise one or more representations of protein-protein interfaces between the first protein building block and the second protein building block that are energetically favorable to drive self-assembly of the protein building blocks comprising the modified amino acid sequences to the docked configuration using the computing device; and   generating an output of the computing device that is based on at least one representation of the group consisting of: a representation of the docked configuration, at least one representation of the plurality of representations of the protein-protein interfaces, and at least one representation of the representations of the first protein building block and the representations of the second protein building block having modified amino acid sequences.   
     
     
         9 . A self-assembling symmetric protein nanostructure comprising two components,
 wherein the first component comprises a multimer of a first protein and the second component comprises a multimer of second protein,   wherein the first and second protein each comprise one or more substitutions at interface positions relative to naturally occurring proteins, and   wherein the two components self-assemble with point group symmetry.   
     
     
         10 . The nanostructure of  claim 9 , wherein the first component comprises a pentamer of the first protein and the second component comprises a trimer of the second protein. 
     
     
         11 . The nanostructure of  claim 10 , wherein the nanostructure comprises 12 pentameric components and 20 trimeric components. 
     
     
         12 . The nanostructure of  claim 11 , wherein the nanostructure has an 153 architecture. 
     
     
         13 . The nanostructure of  claim 9 , wherein the first component comprises a tetramer of the first protein and the second component comprises a dimer of the second protein. 
     
     
         14 . The nanostructure of  claim 9 , wherein the first protein comprises one or more amino acid substitutions at interface residues that contact the second protein, and/or the second protein comprises one or more amino acid substitutions at interface residues that contact the first protein. 
     
     
         15 . A method of generating a nanostructure, the method comprising:
 docking, via computational modeling, a representation of a first multimer comprising a first protein and a representation of a second multimer comprising a second protein to form a representation of a symmetric protein nanostructure comprising two components with point group symmetry;   identifying one or more interface residues in an interface between the first protein and the second protein in the representation of the nanostructure;   modeling substitutions of the one or more interface residues to identify substitutions predicted to stabilize the interface between the first protein and the second protein; and   outputting sequences for the first and second protein modified to comprise the identified substitutions in interface residues.   
     
     
         16 . The method of  claim 15 , further comprising producing the nanostructure by expressing, in a host cell, a nucleic acid encoding the first and second protein modified to comprise the identified substitutions in interface residues. 
     
     
         17 . The method of  claim 15 , further comprising producing the nanostructure by:
 expressing, in a first host cell, the first protein modified to comprise the identified substitutions in interface residues;   expressing, in a second host cell, the second protein modified to comprise the identified substitutions in interface residues; and   contacting the first and second proteins, thereby causing the first and second proteins to self-assemble into the nanostructure.   
     
     
         18 . A polynucleotide comprising sequences encoding a first protein and/or a second protein, wherein the first and/or second proteins form first and second multimers that self-assemble to form a nanostructure with point group symmetry. 
     
     
         19 . A self-assembling symmetric protein nanostructure comprising a first component comprising a plurality of first proteins and a second component comprising a plurality of second proteins,
 wherein the first protein and the second protein comprise proteins selected from the following pairs of first and second proteins:   (a) T32-28A (SEQ ID NO: 11) and T32-28B (SEQ ID NO: 12);   (b) T33-09A (SEQ ID NO: 13) and T33-09B (SEQ ID NO: 14);   (c) T33-15A (SEQ ID NO: 15) and T33-15B (SEQ ID NO: 16);   (d) T33-21A (SEQ ID NO: 17) and T33-21B (SEQ ID NO: 18);   (e) T33-28A (SEQ ID NO: 19) and T33-28B (SEQ ID NO: 20);   (f) T32-28A (SEQ ID NO: 21) and T32-28B (SEQ ID NO: 22);   (g) T33-09A (SEQ ID NO: 23) and T33-09B (SEQ ID NO: 24);   (h) T33-15A (SEQ ID NO: 25) and T33-15B (SEQ ID NO: 26);   (i) T33-21A (SEQ ID NO: 27) and T33-21B (SEQ ID NO: 28);   (j) T33-28A (SEQ ID NO: 29) and T33-28B (SEQ ID NO: 30);   (k) T32-28A (SEQ ID NO: 31) and T32-28B (SEQ ID NO: 32);   (l) T33-09A (SEQ ID NO: 33) and T33-09B (SEQ ID NO: 34);   (m) T33-15A (SEQ ID NO: 35) and T33-15B (SEQ ID NO: 36);   (n) T33-21A (SEQ ID NO: 37) and T33-21B (SEQ ID NO: 38); and   (o) T33-28A (SEQ ID NO: 39) and T33-28B (SEQ ID NO: 40).   
     
     
         20 . A polynucleotide encoding a first protein and/or a second protein selected from the following pairs of first and second proteins:
 (a) T32-28A (SEQ ID NO: 11) and T32-28B (SEQ ID NO: 12);   (b) T33-09A (SEQ ID NO: 13) and T33-09B (SEQ ID NO: 14);   (c) T33-15A (SEQ ID NO: 15) and T33-15B (SEQ ID NO: 16);   (d) T33-21A (SEQ ID NO: 17) and T33-21B (SEQ ID NO: 18);   (e) T33-28A (SEQ ID NO: 19) and T33-28B (SEQ ID NO: 20);   (f) T32-28A (SEQ ID NO: 21) and T32-28B (SEQ ID NO: 22);   (g) T33-09A (SEQ ID NO: 23) and T33-09B (SEQ ID NO: 24);   (h) T33-15A (SEQ ID NO: 25) and T33-15B (SEQ ID NO: 26);   (i) T33-21A (SEQ ID NO: 27) and T33-21B (SEQ ID NO: 28);   (j) T33-28A (SEQ ID NO: 29) and T33-28B (SEQ ID NO: 30);   (k) T32-28A (SEQ ID NO: 31) and T32-28B (SEQ ID NO: 32);   (l) T33-09A (SEQ ID NO: 33) and T33-09B (SEQ ID NO: 34);   (m) T33-15A (SEQ ID NO: 35) and T33-15B (SEQ ID NO: 36);   (n) T33-21A (SEQ ID NO: 37) and T33-21B (SEQ ID NO: 38); and   (o) T33-28A (SEQ ID NO: 39) and T33-28B (SEQ ID NO: 40).   
     
     
         21 . A method of producing a self-assembling protein nanostructure, the method comprising:
 (a) culturing a host cell under conditions conducive to the expression of:
 (i) a plurality of first proteins that self-interact to form a first multimeric substructure comprising at least one axis of rotational symmetry; 
 (ii) a plurality of second proteins that self-interact to form a second multimeric substructure comprising at least one axis of rotational symmetry;
 wherein multiple copies of the first multimeric substructure and the second multimeric substructure interact with each other at one or more symmetrically repeated, non-natural, non-covalent protein-protein interfaces that orient the first multimeric substructures and the second multimeric substructures such that their symmetry axes are aligned with symmetry axes of the same kind in a designated mathematical symmetry group; and 
 
   (b) recovering the expressed proteins.   
     
     
         22 . A self-assembling multimer comprising a plurality of a non-naturally occurring protein that self-interacts to form a multimeric structure comprising at least one axis of rotational symmetry, wherein the multimeric structure is capable of interacting with another multimeric structure such that their symmetry axes are aligned with symmetry axes of the same kind in a designated mathematical symmetry group. 
     
     
         23 . The multimer of  claim 22 , wherein the multimeric structure is dimeric, trimeric, tetrameric or pentameric. 
     
     
         24 . The multimer of  claim 22 , wherein the designated mathematical symmetry group is tetrahedral, octahedral or icosahedral. 
     
     
         25 . The multimer of  claim 22 , wherein the multimer produces a protein nanostructure comprising one or two components. 
     
     
         26 . The multimer of  claim 22 , wherein the protein comprises one or more substitutions at interface positions relative to a naturally occurring protein listed in Table 1 or 2. 
     
     
         27 . The multimer of  claim 22 , wherein the protein comprises an amino acid sequence selected from the group consisting of SEQ ID Nos. 1-44 and 46-50. 
     
     
         28 . A method of generating the multimer of  claim 22 , the method comprising:
 (a) culturing a host cell under conditions conducive to the expression of the protein; and   (b) recovering the expressed protein.   
     
     
         29 . A non-naturally occurring protein comprising an amino acid sequence selected from the group consisting of SEQ ID Nos. 1-44 and 46-50. 
     
     
         30 . A multimeric protein assembly comprising a plurality of a protein of  claim 29 . 
     
     
         31 . A method of producing a non-naturally occurring protein of  claim 29 , the method comprising:
 culturing a host cell under conditions conducive to the expression of the protein; and   recovering the expressed protein.   
     
     
         32 . A method of producing a multimeric protein assembly of  claim 30 , the method comprising:
 culturing a host cell under conditions conducive to the expression of the protein; and   recovering the expressed protein.   
     
     
         33 . A method of producing a nanostructure, wherein the nanostructure comprises self-assembling, non-naturally occurring proteins with amino acid sequences identified by:
 docking, via computational modeling, a representation of a first multimer comprising a first protein and a representation of a second multimer comprising a second protein to form a representation of a symmetric protein nanostructure comprising two components with point group symmetry;   identifying one or more interface residues in an interface between the first protein and the second protein in the representation of the nanostructure;   modeling substitutions of the one or more interface residues to identify substitutions predicted to stabilize the interface between the first protein and the second protein; and   outputting sequences for the first and second protein modified to comprise the identified substitutions in interface residues.   
     
     
         34 . A nanostructure produced by a method comprising:
 docking, via computational modeling, a representation of a first multimer comprising a first protein and a representation of a second multimer comprising a second protein to form a representation of a symmetric protein nanostructure comprising two components with point group symmetry;   identifying one or more interface residues in an interface between the first protein and the second protein in the representation of the nanostructure;   modeling substitutions of the one or more interface residues to identify substitutions predicted to stabilize the interface between the first protein and the second protein; and   outputting sequences for the first and second protein modified to comprise the identified substitutions in interface residues.