The Bacteriorhodopsin Carboxyl-Terminus Contributes to Proton Recruitment and Protein Stability

• Published in: Biochemistry (Easton) 2009-02, Vol.48 (5), p.1112-1122
• Main Authors: Turner, George J, Chittiboyina, Shirisha, Pohren, Lauren, Hines, Kirk G, Correia, John J, Mitchell, Drake C
• Format: Article
• Language: English
• Subjects: Amino Acid Sequence; Bacteria; Bacteriorhodopsins - chemistry; Bacteriorhodopsins - physiology; Base Sequence; Cytoplasm - chemistry; Cytoplasm - metabolism; Escherichia coli - chemistry; Halobacterium salinarum - chemistry; Lipids - chemistry; Molecular Sequence Data; Peptide Fragments - chemistry; Peptide Fragments - physiology; Protein Stability; Protein Structure, Secondary; Protein Structure, Tertiary; Protons; Static Electricity
• ISSN: 0006-2960; 1520-4995
• DOI: 10.1021/bi801799j

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.