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dc.contributorScholtz, J M.
dc.contributorPace, C N.
dc.creatorFu, Hailong
dc.date.accessioned2010-07-15T00:11:57Z
dc.date.accessioned2010-07-23T21:43:31Z
dc.date.accessioned2017-04-07T19:57:04Z
dc.date.available2010-07-15T00:11:57Z
dc.date.available2010-07-23T21:43:31Z
dc.date.available2017-04-07T19:57:04Z
dc.date.created2009-05
dc.date.issued2010-07-14
dc.identifier.urihttp://hdl.handle.net/1969.1/ETD-TAMU-2009-05-444
dc.description.abstractThe 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.
dc.language.isoeng
dc.subjectconformational stability
dc.subjecthydrophobic effect
dc.subjectthermostable
dc.subjectdenatured state
dc.subjectheat capacity change
dc.subjectbeta turn
dc.titleUNDERSTANDING FORCES THAT CONTRIBUTE TO PROTEIN STABILITY: APPLICATION FOR INCREASING PROTEIN STABILITY
dc.typeBook
dc.typeThesis


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