Folding, Stability, and Physical Properties of the Subunit of Bacterial Luciferase
详细信息 查看全文
- 作者：Brian W. Noland ; Lawrence J. Dangott ; and Thomas O. Baldwin
- 刊名：Biochemistry
- 出版年：1999
- 出版时间：December 7, 1999
- 年：1999
- 卷：38
- 期：49
- 页码：16136 - 16145
- 全文大小：223K
- 年卷期：v.38,no.49(December 7, 1999)
- ISSN：1520-4995
文摘
Bacterial luciferase is a heterodimeric (
) enzyme composed of homologous subunits. Whenthe Vibrio harveyi luxA gene is expressed in Escherichia coli, the subunit accumulates to high levels.The subunit has a well-defined near-UV circular dichroism spectrum and a higher intrinsic fluorescencethan the heterodimer, demonstrating fluorescence quenching in the enzyme which is reduced in the freesubunit [Sinclair, J. F., Waddle, J. J., Waddill, W. F., and Baldwin, T. O. (1993) Biochemistry 32, 5036-5044]. Analytical ultracentrifugation of the subunit has revealed a reversible monomer to dimerequilibrium with a dissociation constant of 14.9 ± 4.0 
M at 18 
C in 50 mM phosphate and 100 mMNaCl, pH 7.0. The subunit unfolded and refolded reversibly in urea-containing buffers by a three-statemechanism. The first transition occurred over the range of 0-2 M urea with an associated free-energychange of 2.24 ± 0.25 kcal/mol at 18 C in 50 mM phosphate buffer, pH 7.0. The second, occurringbetween 2.5 and 3.5 M urea, comprised a cooperative transition with a free-energy change of 6.50 ± 0.75kcal/mol. The intermediate species, populated maximally at ca. 2 M urea, has defined near-UV circulardichroism spectral properties distinct from either the native or the denatured states. The intrinsic fluorescenceof the intermediate suggested that, although the quantum yield had decreased, the tryptophanyl residuesremained largely buried. The far-UV circular dichroism spectrum of the intermediate indicated that it hadlost ca. 40% of its native secondary structure. N-Terminal sequencing of the products of limited proteolysisof the intermediate showed that the C-terminal region of the subunit became protease labile over theurea concentration range at which the intermediate was maximally populated. These observations haveled us to propose an unfolding model in which the first transition is the unfolding of a C-terminal subdomainand the second transition represents the unfolding of a more stable N-terminal subdomain. Comparisonof the structural properties of the unfolding intermediate using spectroscopic probes and limited proteolysisof the subunit with those of the heterodimer suggested that the unfolding pathway of the subunitis the same, whether it is in the form of the free subunit or in the heterodimer.
) enzyme composed of homologous subunits. Whenthe Vibrio harveyi luxA gene is expressed in Escherichia coli, the subunit accumulates to high levels.The subunit has a well-defined near-UV circular dichroism spectrum and a higher intrinsic fluorescencethan the heterodimer, demonstrating fluorescence quenching in the enzyme which is reduced in the freesubunit [Sinclair, J. F., Waddle, J. J., Waddill, W. F., and Baldwin, T. O. (1993) Biochemistry 32, 5036-5044]. Analytical ultracentrifugation of the subunit has revealed a reversible monomer to dimerequilibrium with a dissociation constant of 14.9 ± 4.0 M at 18 C in 50 mM phosphate and 100 mMNaCl, pH 7.0. The subunit unfolded and refolded reversibly in urea-containing buffers by a three-statemechanism. The first transition occurred over the range of 0-2 M urea with an associated free-energychange of 2.24 ± 0.25 kcal/mol at 18 C in 50 mM phosphate buffer, pH 7.0. The second, occurringbetween 2.5 and 3.5 M urea, comprised a cooperative transition with a free-energy change of 6.50 ± 0.75kcal/mol. The intermediate species, populated maximally at ca. 2 M urea, has defined near-UV circulardichroism spectral properties distinct from either the native or the denatured states. The intrinsic fluorescenceof the intermediate suggested that, although the quantum yield had decreased, the tryptophanyl residuesremained largely buried. The far-UV circular dichroism spectrum of the intermediate indicated that it hadlost ca. 40% of its native secondary structure. N-Terminal sequencing of the products of limited proteolysisof the intermediate showed that the C-terminal region of the subunit became protease labile over theurea concentration range at which the intermediate was maximally populated. These observations haveled us to propose an unfolding model in which the first transition is the unfolding of a C-terminal subdomainand the second transition represents the unfolding of a more stable N-terminal subdomain. Comparisonof the structural properties of the unfolding intermediate using spectroscopic probes and limited proteolysisof the subunit with those of the heterodimer suggested that the unfolding pathway of the subunitis the same, whether it is in the form of the free subunit or in the heterodimer.