Table 1.

An overview of suggested features for internal thermostability, selected from structural studies of homologues, along with some development approaches to introduce thermostability, and development of thermostable proteins.

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	Disulfide bridges; Hydrogen bonds; Hydrophobic interactions; Aromatic interactions; Ion-pair networks (charged residues); Docking of loose ends	[18–24]
Stabilising interactions between domains/subunits	Oligomer formation via e.g. ion pair networks	[17, 19, 25]
Dense packing	Increase core hydrophobicit;, Fill cavities. Not a generally applicable feature as shown by Karshikoff & Ladenstein [21]	[19]
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.	[17, 24]
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	[27]
Improving activity at selected pH values	Directed evolution	[28]
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.	[29]
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]