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.

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]