Interfacial proteins are water-soluble, interfacial enzymes or regulatory macromolecules that bind transiently to the membrane surface during the course of the catalytic reaction or signaling event. The structures of the water-soluble state are often readily available to high resolution in a routine manner. However, characterizing the conformation of the active protein complex at the membrane interface is a major challenge in structural biology. Current methods rely primarily on the introduction of large, potentially structure altering, probes for EPR or fluorescence analysis, or detection of local structure by solid-state NMR spectroscopy. Moreover, they require construction of dozens of individual site-directed mutants for labeling followed by individual analysis. We are developing membrane-impermeable, isotope-coded labeling reagents that will elucidate interfacial protein binding to the lipid membrane using mass spectrometric methodology. The power of the mass spectrometric method is that it will ultimately allow rapid monitoring of changes in protein conformation under different conditions, e.g., upon addition of drug or regulatory proteins, with small amounts of protein. We are developing this methodology with cholesterol oxidase from Streptomyces because it is easily heterologously expressed and is very stable and high resolution (0.9 Å) X-ray crystal structures of the solution form of the enzyme are available.

Our method to determine the protein-membrane contact interface relies on the differential reactivity of cysteine thiols that are in contact with the membrane versus those that are solvent exposed. We have synthesized membrane impermeable cysteine-labeling reagents optimized for labeling analysis by mass spectrometry. These reagents include heavy isotope labels for direct determination of protection ratios as well as basic moieties that enable ionization in the mass spectrometer. Our method allows determination of membrane contact at several sites simultaneously. We are using these reagents to probe whether residues are in membrane contact or not. Once completed, we will utilize partially membrane impermeable reagents, as well as knowledge gained about the variability of protection factors with membrane impermeable reagents, to determine the depth of the residues in the membrane.