Methods of making polypeptides with non-standard amino acids using genomically recoded organisms
A method of making a polypeptide including at least one covalent bond between a pair of reactive side chains of corresponding amino acids, wherein the covalent bond is insensitive to reduction is provided including genetically modifying a genomically recoded organism to express a corresponding synthetase, tRNA or synthetase/tRNA pair for translating mRNA encoding the corresponding amino acids having the reactive side chains into the polypeptide and to express the polypeptide including the at least one pair of the reactive side chains wherein the reactive side chains are oriented near one another when the expressed polypeptide is in a folded configuration, wherein the reactive side chains react to form the covalent bond that is insensitive to reduction.
1. A method of in silico polypeptide design comprising
identifying a target polypeptide by using a geometric matching algorithm to scan a database of folded wild type protein structures for backbone conformations having target amino acid pair geometries to accommodate geometry of at least one covalent bond between a pair of reactive side chains of corresponding amino acids while substantially maintaining the three-dimensional folded structure of the target polypeptide,
substituting in silico the target amino acid pair with the pair of corresponding amino acids having the pair of reactive side chains to create a modified target polypeptide,
determining a difference in geometry between the three-dimensional folded structure of the target polypeptide and a three-dimensional folded structure of the modified target polypeptide using a geometric matching algorithm or protein design simulation, and
substituting or altering in silico neighboring native amino acids or other native amino acid conformations with substitute amino acids to reduce the difference between the geometry of the three-dimensional folded structure of the target polypeptide and the three-dimensional folded structure of the modified target polypeptide, so as to optimize a reaction distance and orientation to allow the pair of reactive side chains to react under protein folding.
2. The method of claim 1 wherein the corresponding amino acids are selenocysteine.
3. The method of claim 1 wherein the corresponding amino acids are cysteine.
4. The method of claim 1 wherein the pair of reactive side chains include —SeH/—SeH.
5. The method of claim 1 wherein the pair of reactive side chains include —SeH/—SH.
6. The method of claim 1 wherein the pair of reactive side chains include selenol/selenol.
7. The method of claim 1 wherein the pair of reactive side chains include selenol/thiol, selenol/vinyl, tetrazine/strained alkene, or boronate/saccharide.
8. The method of claim 1 further comprising
computer modeling of the three-dimensional folded structure of the target polypeptide in the native environment.
9. The method of claim 1 wherein the target polypeptide has an activity.
10. The method of claim 1 wherein the identified target amino acid pair is replaced by a pair of non-standard amino acids.
11. The method of claim 1 wherein the step of analyzing the three-dimensional folded structure of the target polypeptide to identify target amino acid pair geometries to accommodate geometry of at least one covalent bond between a pair of reactive side chains of corresponding amino acids comprises identifying the target amino acid pair geometries that accommodate geometric conformations of azide and alkyne side-chains forming a triazole ring.