Structure of the peripheral arm of a minimalistic respiratory complex I.

Respiratory complex I drives proton translocation across energy-transducing membranes by NADH oxidation coupled with (ubi)quinone reduction. In humans, its dysfunction is associated with neurodegenerative diseases. The Escherichia coli complex represents the structural minimal form of an energy-converting NADH:ubiquinone oxidoreductase. Here, we report the structure of the peripheral arm of the E. coli complex I consisting of six subunits, the FMN cofactor, and nine iron-sulfur clusters at 2.7 Å resolution obtained by cryo electron microscopy. While the cofactors are in equivalent positions as in the complex from other species, individual subunits are adapted to the absence of supernumerary proteins to guarantee structural stability. The catalytically important subunits NuoC and D are fused resulting in a specific architecture of functional importance. Striking features of the E. coli complex are scrutinized by mutagenesis and biochemical characterization of the variants. Moreover, the arrangement of the subunits sheds light on the unknown assembly of the complex.

Monoclonal antibody 4B1 influences the pKa of Glu325 in lactose permease (LacY) from Escherichia coli: evidence from SEIRAS.

The monoclonal antibody 4B1 binds to a conformational epitope on the periplasmic side of lactose permease (LacY) of Escherichia coli and inhibits H+ /lactose symport and lactose efflux under nonenergized conditions. At the same time, ligand binding and translocation reactions that do not involve net H+ translocation remain unaffected by 4B1. In this study, surface-enhanced infrared absorption spectroscopy applied to the immobilized LacY was used to study the pH-dependent changes in LacY and to access in situ the effect of the 4B1 antibody on the pKa of Glu325, the primary functional H+ -binding site in LacY. A small shift of the pK value from 10.5 to 9.5 was identified that can be corroborated with the inactivation of LacY upon 4B1 binding.

Following the Chemical Immobilization of Membrane Proteins on Plasmonic Nanoantennas Using Infrared Spectroscopy.

Plasmonic nanoantennas are promising sensing platforms for detecting chemical and biological molecules in the infrared region. However, integrating fragile biological molecules such as proteins on plasmonic nanoantennas is an essential requirement in the detection procedure. It is crucial to preserve the structural integrity and functionality of proteins while attaching them. In this study, we attached lactose permease, a large membrane protein, onto plasmonic nanoantennas by means of the nickel-nitrile triacetic acid immobilization technique. We followed the individual steps of the immobilization procedure for different lengths of the nanoantennas. The impact of varying the length of the nanoantennas on the shape of the vibrational signal of the chemical layers and on the protein spectrum was studied. We showed that these large proteins are successfully attached onto the nanoantennas, while the chemical spectra of the immobilization monolayers show a shape deformation which is an effect of the coupling between the vibrational mode and the plasmonic resonance.

Visualizing the movement of the amphipathic helix in the respiratory complex I using a nitrile infrared probe and SEIRAS.

Conformational movements play an important role in enzyme catalysis. Respiratory complex I, an L-shaped enzyme, connects electron transfer from NADH to ubiquinone in its peripheral arm with proton translocation through its membrane arm by a coupling mechanism still under debate. The amphipathic helix across the membrane arm represents a unique structural feature. Here, we demonstrate a new way to study conformational changes by introducing a small and highly flexible nitrile infrared (IR) label to this helix to visualize movement with surface-enhanced IR absorption spectroscopy. We find that labeled residues K551CL and Y590CL move to a more hydrophobic environment upon NADH reduction of the enzyme, likely as a response to the reorganization of the antiporter-like subunits in the membrane arm.

Arg302 governs the pKa of Glu325 in LacY.

Lactose permease is a paradigm for the major facilitator superfamily, the largest family of ion-coupled membrane transport proteins known at present. LacY carries out the coupled stoichiometric symport of a galactoside with an H+, using the free energy released from downhill translocation of H+ to drive accumulation of galactosides against a concentration gradient. In neutrophilic Escherichia coli, internal pH is kept at ∼7.6 over the physiological range, but the apparent pK (pKapp) for galactoside binding is 10.5. Surface-enhanced infrared absorption spectroscopy (SEIRAS) demonstrates that the high pKa is due to Glu325 (helix X), which must be protonated for LacY to bind galactoside effectively. Deprotonation is also obligatory for turnover, however. Here, we utilize SEIRAS to study the effect of mutating residues in the immediate vicinity of Glu325 on its pKa The results are consistent with the idea that Arg302 (helix IX) is important for deprotonation of Glu325.