xiv, 82 p., ill. (some col.) / Cellular processes are carried out by proteins, which often utilize conformational changes for function. In theory, conformational changes can be harnessed to promote, prevent or monitor cellular processes. Such changes in protein active centers require perturbations through interactions with other proteins, small molecules or through energy input into the system, for example light. The work presented incorporates rational design and crystallographic elucidation of two-state conformational changes in two proteins, green fluorescent protein (GFP) and malate synthase (MS).
GFP indicators were previously developed to quantitate the thiol/disulfide redox status within cells. Cysteine residues were introduced in close proximity on the surface of GFP and allow the formation of a disulfide bond. The indicators provide a fluorescent readout of the ambient thiol/disulfide equilibrium, however thermodynamic studies showed the resulting thiol/disulfide to be unusually stable (-287 mV) in comparison to the cellular redox buffer glutathione (-240 mV). In order to produce a family of redox indicators suitable for use in less reducing environments, amino acids were inserted near the introduced cysteine pair in order to destabilize the disulfide. The resulting family of redox indicators, termed roGFP-iX, exhibit midpoint potentials in the more desirable range of -229 to -246 mV. Crystallographic analysis indicates that roGFP-iX indicators undergo much larger two-state conformational changes than the original indicators. Surprisingly, a cis-peptide was discovered between the cysteine and the inserted residue which in combination with the conformational changes helps to explain the reduced stability of the disulfide.
Malate synthase is an important virulence factor for certain microbes and carries out the Claisen condensation between glyoxylate and acctyl-CoA to produce malate. Crystal structures of Mycobacterium tuberculosis and Escherichia coli malate synthase isoform G had previously been determined with substrates or products bound. To determine the conformational changes necessary for substrate binding and product release, crystal structures of Escherichia coli malate synthase isoform A were determined in both the apo and acetyl-CoA/inhibitor bound forms. The crystallographic models revealed two-state conformational changes in the part of the active-site loop necessary for substrate binding, which has important implications for drug design.
This dissertation includes my unpublished co-authored materials. / Adviser: S. James Remington
| Identifer | oai:union.ndltd.org:uoregon.edu/oai:scholarsbank.uoregon.edu:1794/6198 |
| Date | 12 1900 |
| Creators | Lohman, Jeremy R., 1981- |
| Publisher | University of Oregon |
| Source Sets | University of Oregon |
| Language | en_US |
| Detected Language | English |
| Type | Thesis |
| Format | 6002950 bytes, 58695 bytes, application/pdf, application/pdf |
| Relation | University of Oregon theses, Dept. of Chemistry, 2007, Ph. D. |
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