Research

Caveolin-1 is 178 amino acid (~22 kD) primarily helical integral membrane protein that is most notably involved in the generation of plasma membrane invaginations known as caveolae.  It has an atypical topological disposition in the membrane, where the N- and C- termini both reside in the cytoplasm as a result of putative re-entrant intramembrane loop structure.  It has been proposed that this unusual mode of membrane interaction may provide an architectural driving force for caveolae formation.

Deciphering the exact topology of caveolin-1 within a phospholipid bilayer (penetration depth, entrance/exit point into the bilayer, helical orientation and tilt) and molecular determinants which alter the topology (i.e., the presence of specific lipids, role of specific amino acids) are major projects in the Root lab.  In particular, we are using fluorescence spectroscopy to analyze the aqueous accessibility of residues within the membrane interacting domain of the protein using tryptophan and cysteine scanning methodologies.  Ultimately, this work will be used in conjunction with existing and developing structural models to better understand caveolin structure-function relationships.  

Microbial esterases are a highly desirable tool for numerous biosynthetic and biotechnological applications requiring ester bond cleavage. Once identified, microbial esterases are often produced recombinantly in Escherichia coli to enhance yield and ease of purification. In this study a polyhistidine-tagged SGNH esterase gene (AaSGNH1), originating from the cyanobacterium Aphanizomenon flos-aquae, was cloned into an over-expression plasmid and expressed in BL21(DE3) cells. The recombinant esterase enzyme was produced as inactive inclusion bodies which were insoluble in 8 M urea but readily solubilized by the detergent Empigen BB®. Crucially, the procurement of active enzyme required controlled removal of detergent during column chromatography and dialysis steps. The refolded esterase was characterized with respect to its ability to catalyze the cleavage of p-nitrophenol esters of different chain lengths (C2, C8, C16). In addition, the temperature and pH optima were determined and it was found that the enzyme was most active at low temperatures (5-15 °C) and under alkaline conditions (pH 8-10). It was found that the kinetic properties of AaSGNH1 were remarkably similar to other SGNH esterases described thereby validating that the protein was effectively refolded. Overall, this study provides a simple strategy for isolating cold-active recombinant esterase enzyme when expressed as inclusion bodies.  A manuscript detailing this work, all carried out by CUP students, can be found on the publications page.

Lipase enzymes (triacylglycerol acylhydrolases, EC 3.1.1.3) are emerging as a powerful tool for catalysis at the aqueous-lipid interface.  In particular, lipase sourced from thermophilic organisms may hold promise in catalyzing transesterification reactions between methanol and fatty acids due to the requirement of a robust enzyme which can function in non-native conditions with higher organic solvent content.  In addition, thermophilic lipase serve as a template by which rational or randomized mutations can be introduced in order to further understand structural contributions to thermostability, substrate selectivity, and rate enhancement.

Currently, we have over-expressed (in E. coli), purified, and kinetically characterized a thermophilic lipase native to Sphaerobacter thermophilius (ST), a bacterium isolated from thermally treated sewage.  We have found that the ST lipase is operational over a wide range of temperatures with a maximum activity displayed at ~60° C and a preference for medium chain fatty acyl substrates.  In addition to being thermophilic, we have found that it is also robust to alkaline conditions, showing a plateau in optimal activity in the pH 8-11 range.  We have also shown that the immobilization of the lipase on silica-based wide-pore ion -exchange resin has allowed for repeated use in organic solvents without significant loss in activity and demonstrates improved storage stability.  A manuscript detailing this work and co-authored by Commonwealth University of Pennsylvania students in in preparation.