Cardiolipin enhances the enzymatic activity of cytochrome bd and cytochrome bo3 solubilized in dodecyl-maltoside

Cardiolipin (CL) is a lipid that is found in the membranes of bacteria and the inner membranes of mitochondria. CL can increase the activity of integral membrane proteins, in particular components of respiratory pathways. We here report that CL activated detergent-solubilized cytochrome bd, a terminal oxidase from Escherichia coli. CL enhanced the oxygen consumption activity ~ twofold and decreased the apparent KM value for ubiquinol-1 as substrate from 95 µM to 35 µM. Activation by CL was also observed for cytochrome bd from two Gram-positive species, Geobacillus thermodenitrificans and Corynebacterium glutamicum, and for cytochrome bo3 from E. coli. Taken together, CL can enhance the activity of detergent-solubilized cytochrome bd and cytochrome bo3.

Engineering CGTase to improve synthesis of alkyl glycosides

Alkyl glycoside surfactants with elongated carbohydrate chains are useful in different applications due to their improved biocompatibility. Cyclodextrin glucanotransferases can catalyze the elongation process through the coupling reaction. However, due to the presence of a hydrophobic tail, the interaction between an alkyl glycoside acceptor and the active site residues is weaker than the interaction with maltooligosaccharides at the corresponding site. Here we report the mutations of F197, G263 and E266 near the acceptor subsites in the CGTase CspCGT13 from Carboxydocella sp. The results showed that substitutions of both F197 and G263 were important for the binding of acceptor substrate dodecyl maltoside during coupling reaction. The double mutant F197Y/G263A showed enhanced coupling activity and displayed a 2-fold increase of the primary coupling product using γ-cyclodextrin as donor when compared to wildtype CspCGT13. Disproportionation activity was also reduced, which was also the case for another double mutant (F197Y/E266A) that however not showed the corresponding increase in coupling. A triple mutant F197Y/G263A/E266A maintained the increase in primary coupling product (1.8-fold increase) using dodecyl maltoside as acceptor, but disproportionation was approximately at the same level as in the double mutants. In addition, hydrolysis of starch was slightly increased by the F197Y and G263A substitutions, indicating that interactions at both positions influenced the selectivity between glycosyl and alkyl moieties.

Interaction of Cytochrome C Oxidase with Steroid Hormones

Estradiol, testosterone and other steroid hormones inhibit cytochrome c oxidase (CcO) purified from bovine heart. The inhibition is strongly dependent on concentration of dodecyl-maltoside (DM) in the assay. The plots of Ki vs [DM] are linear for both estradiol and testosterone which may indicate an 1:1 stoichiometry competition between the hormones and the detergent. Binding of estradiol, but not of testosterone, brings about spectral shift of the oxidized CcO consistent with an effect on heme a33+. We presume that the hormones bind to CcO at the bile acid binding site described by Ferguson-Miller and collaborators. Estradiol is shown to inhibit intraprotein electron transfer between hemes a and a3. Notably, neither estradiol nor testosterone suppresses the peroxidase activity of CcO. Such a specific mode of action indicates that inhibition of CcO activity by the hormones is associated with impairing proton transfer via the K-proton channel.

Structural Analysis of a Filamentous Chaperonin from Sulfolobus solfataricus

SUMMARY/AbstractChaperonins are biomolecular complexes that assist protein folding. Thermophilic Factor 55 (TF55) is a group II chaperonin found in the archaeal genus Sulfolobus and which undergoes changes in modular subunit composition in a temperature-dependent manner. TF55 can form filamentous assemblies that may be a component of the archaeal cytoskeleton or sequester inactive chaperonin. Using cryo-electron microscopy, we have determined the structure of the β-only complex of S. solfataricus TF55 complexes to 3.6 Å resolution and its filamentous form to 5.2 Å resolution. Filament formation can be induced when the protein is enriched in solution or in the presence of the detergent dodecyl maltoside. Helical protrusions in the apical domain facilitate end-on-end interactions in the filamentous state. Our findings establish the molecular basis for forming chaperonin filaments in Sulfolobus and may suggest how filament formation could function as a cold-shock response and provides a background for generating tuneable protein nanowires.

Release of Pharmaceutical Peptides in an Aggregated State: Using Fibrillar Polymorphism to Modulate Release Levels

Traditional approaches to achieve sustained delivery of pharmaceutical peptides traditionally use co-excipients (e.g., microspheres and hydrogels). Here, we investigate the release of an amyloidogenic glucagon analogue (3474) from an aggregated state and the influence of surfactants on this process. The formulation of peptide 3474 in dodecyl maltoside (DDM), rhamnolipid (RL), and sophorolipid (SL) led to faster fibrillation. When the aggregates were subjected to multiple cycles of release by repeated resuspension in fresh buffer, the kinetics of the release of soluble peptide 3474 from different surfactant aggregates all followed a simple exponential decay fit, with half-lives of 5–18 min and relatively constant levels of release in each cycle. However, different amounts of peptide are released from different aggregates, ranging from 0.015 mg/mL (3475-buffer) up to 0.03 mg/mL (3474-DDM), with 3474-buffer and 3474-RL in between. In addition to higher release levels, 3474-DDM aggregates showed a different amyloid FTIR structure, compared to 3474-RL and 3474-SL aggregates and a faster rate of degradation by proteinase K. This demonstrates that the stability of organized peptide aggregates can be modulated to achieve differences in release of soluble peptides, thus coupling aggregate polymorphism to differential release profiles. We achieved aggregate polymorphism by the addition of different surfactants, but polymorphism may also be reached through other approaches, including different excipients as well as changes in pH and salinity, providing a versatile handle to control release profiles.

Conformational equilibria of light-activated rhodopsin in nanodiscs

Conformational equilibria of G-protein–coupled receptors (GPCRs) are intimately involved in intracellular signaling. Here conformational substates of the GPCR rhodopsin are investigated in micelles of dodecyl maltoside (DDM) and in phospholipid nanodiscs by monitoring the spatial positions of transmembrane helices 6 and 7 at the cytoplasmic surface using site-directed spin labeling and double electron–electron resonance spectroscopy. The photoactivated receptor in DDM is dominated by one conformation with weak pH dependence. In nanodiscs, however, an ensemble of pH-dependent conformational substates is observed, even at pH 6.0 where the MIIbH+ form defined by proton uptake and optical spectroscopic methods is reported to be the sole species present in native disk membranes. In nanodiscs, the ensemble of substates in the photoactivated receptor spontaneously decays to that characteristic of the inactive state with a lifetime of ∼16 min at 20 °C. Importantly, transducin binding to the activated receptor selects a subset of the ensemble in which multiple substates are apparently retained. The results indicate that in a native-like lipid environment rhodopsin activation is not analogous to a simple binary switch between two defined conformations, but the activated receptor is in equilibrium between multiple conformers that in principle could recognize different binding partners.