The aim of this study is to further our understanding of the forces that contribute
to protein stability and to investigate how site-directed mutagenesis might be used for
increasing protein stability. Eleven proteins ranging from 36 to 370 residues have been
studied here. A 36-residue VHP and a 337-residue VlsE were used as model systems for
studying the contribution of the hydrophobic effect on protein stability. Mutations were
made in both proteins which replaced bulky hydrophobic side chains with smaller ones.
All variants were less stable than their wild-type proteins. For VHP, the destabilizing
effects of mutations were smaller when compared with similar mutations reported in the
literature. For VlsE, a similarity was observed. This different behavior was investigated
and reconciled by the difference in hydrophobicity and cavity modeling for both
proteins. Therefore, the stabilizing mechanism of the hydrophobic effect appears to be
similar for both proteins.
Eight proteins were used as model systems for studying the effects of mutating
non-proline and non-glycine residues to statistically favored proline and glycine residues
in ?-turns. The results suggest that proline mutations generally increase protein stability, provided that the replaced residues are solvent exposed. The glycine mutations,
however, only have a stabilizing effect when the wild-type residues have ?, ? angles in
the L? region of Ramachandran plot. Nevertheless, this strategy still proves to be a
simple and efficient way for increasing protein stability.
Finally, using a combination of eight previously identified stabilizing mutations;
we successfully designed two RNase Sa variants (7S, 8S) that have both much higher
Tms and conformational stabilities than wild-type protein over the entire pH range
studied. Further studies of the heat capacity change upon unfolding (?Cps) for both
proteins and their variants suggest that residual structure may exist in the denatured state
of the 8S variant. An analysis of stability curves for both variants suggests that they
achieve their stabilization through different mechanisms, partly attributed to the different
role of their denatured states. The 7S variants may have a more rigid denatured state and
the 8S variant may have a compact denatured state in comparison with that of wild-type
RNase Sa.
| Identifer | oai:union.ndltd.org:tamu.edu/oai:repository.tamu.edu:1969.1/ETD-TAMU-2009-05-444 |
| Date | 2009 May 1900 |
| Creators | Fu, Hailong |
| Contributors | Scholtz, J M., Pace, C N. |
| Source Sets | Texas A and M University |
| Language | English |
| Detected Language | English |
| Type | Book, Thesis, Electronic Dissertation, text |
| Format | application/pdf |
Page generated in 0.0018 seconds