Functional Studies on Membrane Proteins by Means of H/D Exchange in Infrared: Structural Changes in Na NQR from V. cholerae in the Presence of Lipids.

H/D exchange kinetics at the level of the amide proton in the mid infrared (1700-1500 cm) make it possible to study the conformational flexibility of membrane proteins, independent of size or the presence of detergent or lipids. Slow, medium, and fast exchanging domains are distinguished, which reveal a different accessibility to the solvent. Whereas amide hydrogens undergo rapid exchange with solvent in an open structure, hydrogens experience much slower exchange when involved in H-bonded structures or when sterically inaccessible to the solvent.

Infrared spectroscopic studies on reaction induced conformational changes in the NADH ubiquinone oxidoreductase (complex I).

Redox-dependent conformational changes are currently discussed to be a crucial part of the reaction mechanism of the respiratory complex I. Specialized difference Fourier transform infrared techniques allow the detection of side-chain movements and minute secondary structure changes. For complex I, (1)H/(2)H exchange kinetics of the amide modes revealed a better accessibility of the backbone in the presence of NADH and quinone.

Involvement of Acidic Amino Acid Residues in Zn(2+) Binding to Respiratory Complex I.

Proton transfer across membranes and membrane proteins is a central process in biological systems. Zn(2+) ions are capable of binding to acidic residues, often found within such specific pathways, thereby leading to a blockage. Here we probed Zn(2+) inhibition of the proton-pumping NADH:ubiquinone oxidoreductase from Escherichia coli by means of electrochemically induced FTIR difference spectroscopy.

Inhibition of Escherichia coli respiratory complex I by Zn(2+).

The energy-converting NADH:ubiquinone oxidoreductase, respiratory complex I, couples NADH oxidation and quinone reduction with the translocation of protons across the membrane. Complex I exhibits a unique L shape with a peripheral arm extending in the aqueous phase and a membrane arm embedded in the lipid bilayer. Both arms have a length of ∼180 Å.

Biomimetic environment to study E. coli complex I through surface-enhanced IR absorption spectroscopy.

In this study complex I was immobilized in a biomimetic environment on a gold layer deposited on an ATR-crystal in order to functionally probe the enzyme against substrates and inhibitors via surface-enhanced IR absorption spectroscopy (SEIRAS) and cyclic voltammetry (CV). To achieve this immobilization, two methods based on the generation of a high affinity self-assembled monolayer (SAM) were probed. The first made use of the affinity of Ni-NTA toward a hexahistidine tag that was genetically engineered onto complex I and the second exploited the affinity of the enzyme toward its natural substrate NADH.