The Bacteriorhodopsin Carboxyl-Terminus Contributes to Proton Recruitment and Protein Stability
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- 作者：George J. Turner ; Shirisha Chittiboyina ; Lauren Pohren ; Kirk G. Hines ; John J. Correia ; Drake C. Mitchell
- 刊名：Biochemistry
- 出版年：2009
- 出版时间：February 10, 2009
- 年：2009
- 卷：48
- 期：5
- 页码：1112-1122
- 全文大小：308K
- 年卷期：v.48,no.5(February 10, 2009)
- ISSN：1520-4995
文摘
We examined functional and structural roles for the bacteriorhodopsin (bR) carboxyl-terminus. The extramembranous and intracellular carboxyl-terminus was deleted by insertion of premature translation stop codons. Deletion of the carboxyl-terminus had no effect on purple membrane (PM) lattice dimensions, sheet size, or the electrogenic environment of the ground-state chromophore. Removal of the distal half of the carboxyl-terminus had no effect on light-activated proton pumping, however, truncation of the entire carboxyl-terminus accelerated the rates of M-state decay and proton uptake ∼3.7-fold and severely distorted the kinetics of proton uptake. Differential scanning calorimetry (DSC) and SDS denaturation demonstrated that removal of the carboxyl-terminus decreased protein stability. The DSC melting temperature was lowered by 6 °C and the calorimetric enthalpy reduced by 50% following removal of the carboxyl-terminus. Over the time range of milliseconds to hours at least 3 phases were required to describe the SDS denaturation kinetics for each bR construction. The fastest phases were indistinguishable for all bR’s, and reflected PM solubilization. At pH 7.4, 20 °C, and in 0.3% SDS (w/v) the half-times of bR denaturation were 19.2 min for the wild-type, 12.0 min for the half-truncation and 3.6 min for the full-truncation. Taken together the results of this study suggest that the bR ground state exhibits two “domains” of stability: (1) a core chromophore binding pocket domain that is insensitive to carboxyl-terminal interactions and (2) the surrounding helical bundle whose contributions to protein stability and proton pumping are influenced by long-range interactions with the extramembranous carboxyl-terminus.