Thermodynamic and kinetic studies of the polypeptide folding of bacterial luciferase from Vibrio harveyi : a mutational analysis /
| Main Author: | |
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| Other Authors: | , , |
| Format: | Thesis Book |
| Language: | English |
| Published: |
1994.
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| Subjects: | |
| Online Access: | Link to OAKTrust copy Link to ProQuest copy http://proxy.library.tamu.edu/login?url=http://proquest.umi.com/pqdweb?did=741326061&sid=1&Fmt=2&clientId=2945&RQT=309&VName=PQD |
| Abstract: | Bacterial luciferase is a heterodimeric enzyme which catalyzes the reaction of FMNH2 , O2 , and an aliphatic aldehyde to yield the carboxylic acid and an excited flavin which emits blue-green light upon return to ground state. The two subunits of the luciferase from Vibrio harveyi, c l and p, have molecular weights of 40,108 and 36,349, respectively; the single active center resides primarily, if not exclusively, on the a subunit. Conditions have been established such that bacteria! luciferase can be unfolded in urea-containing 50 mM phosphate buffer, pH 7.0, and refolded by dilution of the urea with final luciferase concentrations of 5-25 jig/mL These measurements demonstrate that the equilibrium unfolding of bacterial luciferase proceeds through a well-populated inactive heterodimeric species that appears to be partially unfolded, and are consistent with the observation of Ziegler et al. (1993) of an inactive heterodimeric kinetic intermediate that forms from the individual subunits and precedes the active heterodimeric enzyme on the refolding pathway. Mutants of luciferase with substitutions for aspartate at position 313 of the p subunit had similar conformational stabilities yet slower refolding rates compared to wild-type luciferase. Studies of luciferase with proline at position {3313 showed the rate of heterodimerization to be approximately 17 fold slower than wild-type luciferase, thereby facilitating partitioning between formation of heterodimer and a stably folded structure of the p subunit. The folded p subunit of PD313P showed circular dichroism properties indicative of a molten globule state. The stable molten globule state of pD313P luciferase did not interact with free a subunit, even after prolonged incubation, indicating that the stable molten globule was not in equilibrium with the heterodimerization-competent conformation. Substitution of each of the eight tryptophans in luciferase demonstrates that multiple tryptophan substitutions should be possible without significantly affecting enzyme stability or activity. In addition, the tryptophan at position a194 is accessible to quenching by iodide in the native state of luciferase, and the tryptophans at a194 and a250 appear to be in the active site of the protein and may be involved in stacking interactions with the flavin substrate. |
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| Item Description: | Vita. "Major subject: Biochemistry." |
| Physical Description: | xvii, 225 leaves : illustrations ; 28 cm |
| Bibliography: | Includes bibliographical references. |