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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.

From: Potential and utilization of thermophiles and thermostable enzymes in biorefining

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]