Characterization of the folding mechanism of the α subunit of bacterial luciferase /
Bacterial luciferase is a heterodimeric (αβ) enzyme composed of homologous subunits. When the Vibrio harveyi luxA gene is expressed in E. coli, the α subunit accumulates to high levels. The α subunit has a well-defined near UV circular dichroism spectrum and a higher intrinsic fluorescence than th...
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| Format: | Thesis Book |
| Language: | English |
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[Place of publication not identified] :
[publisher not identified] ;
2001.
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| Subjects: | |
| Online Access: | http://proxy.library.tamu.edu/login?url=http://proquest.umi.com/pqdweb?did=728908701&sid=1&Fmt=2&clientId=2945&RQT=309&VName=PQD |
| Summary: | Bacterial luciferase is a heterodimeric (αβ) enzyme composed of homologous subunits. When the Vibrio harveyi luxA gene is expressed in E. coli, the α subunit accumulates to high levels. The α subunit has a well-defined near UV circular dichroism spectrum and a higher intrinsic fluorescence than the heterodimer, demonstrating fluorescence quenching in the enzyme which is reduced in the free subunit [Sinclair, J. F., Waddle, J. J., Waddill, W. F. & Baldwin, T. O. (1993) Biochemistry 32, 5036-5044]. Analytical ultracentrifugation of the α subunit has revealed a reversible monomer-to-dimer equilibrium. The α subunit unfolded and refolded reversibly in urea-containing buffers by a three-state mechanism. The first transition occurred over the range of 0-2 M urea and the second occurred between 2.5 and 3.5 M urea. The intrinsic fluorescence and near-UV circular dichroism spectrum of the intermediate suggested that the tryptophan residues are largely buried and the environment of the aromatic residues is distinct from either the native or denatured states. The far-UV circular dichroism spectrum of the intermediate indicated that it had lost ca. 35% of its native secondary structure. N-terminal sequencing of the products of limited proteolysis of the intermediate showed that the C-terminal region of the a subunit became highly protease labile over the urea concentration range at which the intermediate was maximally populated. These observations led to the proposal that the observed three-state unfolding of the α subunit represented the stepwise unfolding of a C-terminal followed by an N-terminal domain. Fragmentation studies, ligand binding studies and pulsed-alkylation mass spectrometry analyses were consistent with this hypothesis. The interaction of the α subunit with the β subunit to form the heterodimer stabilized the α subunit C-terminal folding domain by ca. 2 kcal/mol. The kinetics of unfolding and refolding of the α subunit monitored by fluorescence spectroscopy were multiphasic and involved fast and slow steps. At intermediate urea concentrations the α subunit was slowly converted to a kinetically stable form. This slow process involved the partitioning of the α subunit into at least four kinetically distinct species and was most consistent with a non-sequential process. |
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| Item Description: | Vita. "Major Subject: Biochemistry". |
| Physical Description: | xiv, 133 leaves : illustrations ; 28 cm. Issued also on microfiche from University Microfilm Inc. |
| Bibliography: | Includes bibliographical references (leaves 125-132). |