|Proposed features for internal stabilisation in thermostable proteins||Contributing factors||References|
|Helix stabilisation||Low frequency of Cβ-branched amino acids (e.g. Val, Ile, Thr). Specific amino acids at helical ends (e.g. Pro)||[16, 17]|
|Stabilising interactions in folded protein||
Ion-pair networks (charged residues);
Docking of loose ends
|Stabilising interactions between domains/subunits||Oligomer formation via e.g. ion pair networks||[17, 19, 25]|
Increase core hydrophobicit;, Fill cavities.|
Not a generally applicable feature as shown by Karshikoff & Ladenstein 
|Stable surface-exposed amino acids||Low level of surface amino acids prone to deamidation (e.g. Gln, Asn) or oxidative degradation (e.g. Cys, Met)||[17, 24]|
|Approaches to introduce internal thermostability in mesophilic proteins||Engineering methodology|
|Reducing length of or stabilising surface loops and turns||
Structure-based site directed mutagenesis.|
Promising results reported for:
Loop deletions; Proline-stabilisation of loops;
Docking of loose ends.
|Introduce stabilising interactions||Structure-based site directed mutagenesis. Success reported for introduction of ion-pairs, disulphide bridges, while core packing and helix stabilisation usually do not result in high stability gain.||[17, 24]|
|Activity screen of diversified library at desired temperature||Directed evolution and other random methods utilized successfully in several cases||[24, 26]|
|Approaches to develop thermostable proteins|
|Diversifying specificity||(Structure-based) directed evolution by e.g. oligonucleotide randomisation in active site region, successfully utilized|||
|Improving activity at selected pH values||Directed evolution|||
|Broadening temperature range for activity by introducing flexibility in active site region||
(Structure-based) directed evolution
Patent by Diversa.
Can be made e.g. by oligonucleotide randomisation in active site region.
Saturation mutagenesis at selected positions also used.
|Substitution of surface-exposed amino acids to achieve long term stability||Site directed or saturation mutagenesis at selected positions to reduce Gln, Asn, Cys, Met, suggested||[16, 17]|