Table:
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 evolutionPatent 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]

Convert the figure into Markdown language.

| 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 evolutionPatent 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] |