Charge-charge interactions exist in the denatured state ensemble of ribonuclease Sa : a dissertation /
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| Format: | Thesis Book |
| Language: | English |
| Published: |
[College Station, Tex.] :
[Texas A&M University System Health Science Center],
[2009]
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| Subjects: |
| Abstract: | ABSTRACT: The native conformation of a protein is in thermodynamic equilibrium with the denatured state ensemble (DSE) under physiological conditions. We know a lot about the native state, but there is uncertainty about the structural character of the DSE. Early studies proposed that the DSE had no defined structure. However, the possibility was never completely ruled out. Later studies provided evidence that significant structure exists even in the presence of high denaturant. Current evidence suggests that stable interactions are present in the DSE. DSE interactions may lead to varying amounts of structure. The current study uses sedimentation velocity analytical ultracentrifugation (SV-AUC), protein folding kinetics and fluorescence spectroscopy to investigate interactions in the DSE of several proteins. SV-AUC was utilized to determine the hydrodynamic radius (Rh) of the denatured forms of barnase, ribonuclease T1 and ribonuclease Sa from neutral pH to acidic pH. Changes in Rh suggest alterations in the size and properties of the DSE due to charge-charge interactions. This finding is consistent with pH-dependent m values, which are thought to reflect changes in the structure of the DSE. Protein folding kinetics showed that stabilizing and destabilizing change-reversed variants had distinct kinetic effects. The stabilizing charge-reversed variants of ribonuclease Sa, D25R* and E74R*, refolded and unfolded faster than WT* suggesting that the free energy of the native state and the DSE were decreased. The destabilizing variants, D17R* and E41R* unfolded faster than the wild-type protein (WT*). The refolding rates were similar to WT* suggesting that the destabilizing variants decreased the free energy of the DSE. The interactions caused by the stabilizing and destabilizing mutations persisted in the transition state. Finally, fluorescence spectroscopy provides information about the amount of structure present in unfolded ribonuclease Sa in the absence of denaturant. The fluorescence intensities of several Trp-containing variants were markedly decreased. The wavelengths of maximum intensity were similar to previous values at high denaturant concentrations. We conclude that extensive charge-charge interactions exist in the DSE of barnase, ribonuclease T1, and ribonuclease Sa. The charge-charge network affects the refolding and unfolding kinetics of ribonuclease Sa. |
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| Item Description: | Vita. "Major Subject: Medical Sciences". "Submitted to the Office of Research and Graduate Studies of The Texas A&M University System Health Science Center in partial fulfillment for the requirements for the degree of Doctor of Philosophy May 2009." Approved as to style and content by: J. Martin Scholtz, David N. Brems, C. Nick Pace, David H. Russell. |
| Physical Description: | xi, 134 leaves : illustrations ; 28 cm. |
| Bibliography: | Includes bibliographical references (leaves 125-129). |