Function of Membrane Enzymes in their Native Lipid Environment

The coupling between the complex membrane environment and its affiliated membrane proteins is among the most facinating puzzles of cellular functions. It exemplifies Nature's creative solution to how hydrophobic chemistry can be conducted in a hydrophilic surrounding. s Membrane proteins function in a dynamic material, where the membrane physiochemical conditions (e.g. composition, charge, fluidity, lateral pressure profile, phase separation) govern their reactivity. We study the function of monotopic membrane enzymes. This family of integral membrane proteins resides in only one leaflet of the bilayer and specializes in catalysis of hydrophobic substrates that reside deep within the membrane. They are key enzymes in lipid mediated signaling, steroid synthesis and neurological function and are important therapeutic targets. Members of this protein family employ large hydrophobic surfaces to submerge into the non-polar part of the membrane and access their substrates. The latter requires that they actively modify the structure of the lipid bilayer while at the same time maintain its integrity. We are interested in deciphering how monotopic proteins interface with the membrane and deliver their substrate and products to and from the lipid bilayer. We are also interested in how protein conformational rearrangement assists chemical reactivity, and what unique features arise when such conformationa changes are linked to the bulk environment of cellular membranes.

We study the functional coupling between the integral membrane enzyme, 2,3-oxidosqualene cyclase (OSC) and its lipid environment. OSC is the key protein in the biosynthesis and regulation of cholesterol. It catalyzes the cyclization reaction producing lanosterol, the core skeleton of steroids and hormones. To reach its lipidic substrate, OSC – like all members of the monotopic membrane enzyme family - stably and permanently resides in one leaflet of the bilayer only. Like the other enzymes in this protein family, OSC uses large hydrophobic surfaces to contact the lipid bilayer and utilizes extended hydrophobic channels to shuttle its hydrophobic reactants between its active site and the membrane. Several methods are employed to study the enzymatic reaction of OSC: Several methods are employed in this project: solid-state NMR spectroscopy and fluorescence microscopy and spectroscopy. Our long term goal is to understand the correlation – as mediated by the membrane- between the conformational changes of the protein and the transfer of the substrate and product to and from the lipid bilayer.

Selenoproteins

Selenoproteins are a specialized group of enzymes that contain the reactive amino acid selenocysteine. The presence of selenium as part of the active site creates powerful enzymes whose low redox potential is employed to regulate sulfur based redox pathways. Most selenoproteins, only 25 proteins exist in humans, are oxidoreductases, such as the important redox enzymes thioredoxin reductase and glutathione peroxidase. Those enzymes are cardinal contributors to cellular antioxidative defense and cancer prevention. Functional analysis of selenoproteins is hindered by the absence of a direct method to measure selenium properties, such as bonding, pKa and electronic structure. We develop 77Se solid-state NMR as a versatile spectroscopic method for examining the unique group of selenoproteins, allowing the identification of the active form and local environment of selenium in proteins. The objective is to record selenoproteins redox potential, nucleophilicity and susceptibility to oxidative damage, thereby deducing the general themes by which the protein environment governs these properties.

Current research projects include:

i) Redox potential of selenoproteins
ii) Structure and Function of membrane selenoenzymes iii)
Oxidative damage of thioredoxin-like selenoproteins