Stabilized proteins

A method for making a stabilized protein or fragment thereof comprising:

• (a) selecting one or more residue pairs in a polypeptide chain or chains for cross-linking using one or more criteria, wherein the criteria are based on statistical evaluation of physical, structural, geometrical, or functional measurements made across a sample of structurally, functionally, or both structurally and functionally related proteins, protein complexes or fragments thereof;
• and
• (b) cross-linking the selected residue pairs,
• wherein at least one selected residue is substituted with an amino acid that comprises a reactive side-chain for cross-linking, and the cross-linked protein retains at least one functional activity displayed by the protein in the absence of cross-linking.

A method for identifying a residue pair in a polypeptide chain or chains that, following substitution of at least one selected residue with tyrosine and cross-linking, is least likely to be disruptive of the overall protein structure, the method comprising using one or more statistical criteria selected from the group consisting of statistical filter one through statistical filter eight, wherein each statistical filter selects suitable, or eliminates unsuitable, residue pairs based on statistical evaluation of measurements made across a sample of aligned residues of structurally, functionally, or both structurally and functionally related proteins, protein complexes or fragments thereof, which statistical evaluations of each filter are as follows:

• Filter 1:
• mean, median, or mode of residue pairs' alpha-carbon distance;
• Filter 2:
• standard deviation, variance, maximum, or minimum of residue pairs' alpha-carbon distance;
• Filter 3:
• mean, median, or mode of measurements relating to the three-dimensional orientations of the side-chains of residue pairs;
• Filter 4:
• standard deviation, variance, maximum, or minimum of measurements relating to the three-dimensional orientations of the side-chains of residue pairs;
• Filter 5:
• mean, median, or mode of physical measurements of either of the side-chains of residue pairs;
• Filter 6:
• standard deviation, variance, maximum, or minimum of physical measurements of either of the side-chains of residue pairs;
• Filter 7:
• mean, median, mode, standard deviation, variance, maximum, or minimum of physical measurements of side-chains in three-dimensional or sequence proximity to either of the side-chains of residue pairs;
• and
• Filter 8:
• degree of amino acid conservation or identity of one or both residues of residue pairs.

A method for making a stabilized protein or fragment thereof, comprising:

• (a) selecting residue pairs in a polypeptide chain or chains for and against cross-linking using one or more criteria based on statistical evaluation of physical, structural, geometrical, or functional measurements made across a sample of structurally, functionally, or both structurally and functionally related proteins, protein complexes or fragments thereof;
• and
• (b) cross-linking the residue pairs,
• wherein at least one residue is substituted with an amino acid to prevent cross-linking, and the cross-linked protein retains at least one functional activity displayed by the protein in the absence of cross-linking.

A method for making a stabilized protein or fragment thereof, comprising:

• (a) selecting residue pairs in a polypeptide chain or chains for and against di-tyrosine cross-linking based on statistical evaluation of physical, structural, geometrical, or functional measurements made across a sample of structurally, functionally, or both structurally and functionally related proteins, protein complexes or fragments thereof;
• and
• (b) cross-linking tyrosine residues,
• wherein at least one tyrosine residue is substituted with a non-tyrosine amino acid to prevent cross-linking, and the cross-linked protein retains at least one functional activity displayed by the protein in the absence of cross-linking.