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 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) Structure and function of membrane selenoenzymes
Selenoprotein K (SelK) and S (SelS) are membrane proteins
residing in the endoplasmic reticulum. The function of SelK
is unknown but it was shown to participate in anti-oxidant
defense in vivo, possibly in the Endoplasmic Reticulum
Associated Protein Degradation (ERAD) pathway – a quality
control system responsible for dislocation of misfolded
proteins from the ER for degradation in the cytoplasm. SelS
has been identified as a member of the ERAD machinery and as
a binding partner of SelK. Due to the challenges of
isolating membrane proteins and selenoproteins, the precise
enzymatic function of SelK and S and the specific role of
the rare selenocysteine in this function are still
undetermined. In order to make both enzymes available to
structural and functional experiments, we have developed a
bacterial overexpression and purification strategy to
produce the full length enzyme containing the membrane
spanning segment as well as the reactive selenocysteine.
ii) Redox potential of selenoproteins
The redox potential of
selenoproteins could potentially be considerably low relative
to redox potential of cysteine-containing proteins and overall
range of redox potential utilized in Nature. The redox
potential of only a few artificial selenoproteins has been
determined experimentally, but the existing experimental
evidence suggests that selenoproteins are not endowed with
extreme redox potentials. We study redox potential of human
selenoproteins with a minimal thioredoxin fold, a common class
of selenoproteins involved in signaling. We also study their
susceptibility to oxidative damage.
iii) Biological selenium NMR
We record the electronic environment of the reactive selenium in selenoenzymes’ active sites by solution and solid-state 77Se NMR spectroscopy. 77Se chemical shift are recorded in a series of representative human selenoproteins in which the residues neighboring the selenocysteine are systematically altered. The redox potential and susceptibility of these selenoproteins to modifications by reactive oxygen species will then be determined and correlated to the electronic structure of reactive enzymatic centers. The emerging relationships will explain how the local environment of selenium in selenoproteins fine tunes their chemical reactivity.
The coupling between the complex membrane environment and its affiliated membrane proteins is among the most fascinating 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 conformational 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.