Current Research

Cellular life takes place in a dynamic, heterogeneous, constantly restructuring environment. My research group studies how the chemical reactivity of enzymes is fine tuned and regulated to operate successfully in this fluctuating environment. One central research focus in the Rozovsky lab is the contribution of the membrane properties to the high specificity and rate enhancement of membrane enzymes. A second central theme is the biological role of selenium in proteins that utilize the rare amino acid selenocysteine. Selenoproteins signal the level of cellular oxidative stress and play a central role in regulating redox pathways, thereby helping to limit oxidative stress while maintaining proper cell function. We seek to understand the key differences that set selenoproteins apart from their cysteine-containing homologues.

To experimentally address these diverse questions my lab employs solid and solution-state nuclear magnetic resonance (NMR) spectroscopy as well as a variety of optical microscopy techniques ranging from single molecule total internal reflection fluorescence (TIRF) microscopy to spectral imaging.

Figure 1 A) The structure of a monotopic membrane enzyme (the human oxidosqualene synthase) residing in one leaflet of the lipid bilayer. The active site is linked to the membrane interior via a 15 Å hydrophobic channel, which mediates substrate uptake and product release. The disordered lipid (green) in the channel's entrance (green) illustrates the substrate path from the membrane to the active site. The enzyme's position in the membrane (grey) was inferred from the position of crystallographically observed detergent molecules (orange). B) The surface representation showing the hydrophobic patch that resides in the membrane. A disordered lipid (green) is inside the hydrophobic channel leading to the active site.

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 fascinating puzzles of cellular function. It exemplifies Nature's creative solution to how hydrophobic chemistry can be conducted in a hydrophilic surrounding. 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.

Figure 2 Identification of selenocysteine in a cysteine-selenocysteine redox motif inserted in thioredoxin. The cysteine-selenocysteine redox motif is commonly found in selenoproteins, such as thioredoxin reductase. This (1H-77Se) - Heteronuclear Multiple Bond Coherence (HMBC) spectrum of 77Se enriched protein demonstrates the effectiveness of 77Se NMR in reporting the chemical and electronic environment of selenocysteine in enzymes active sites.

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.