|
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] |
Turner et al. Microbial Cell Factories 2007 6:9 doi:10.1186/1475-2859-6-9 |
||