Grant Ganshaw

A silicone-based controlled-release device for accelerated proteolytic debridement of wounds.

A new device for rapid enzymatic debridement of cutaneous wounds has been developed using a controlled‐release, silicone‐based, dried emulsion. A dehydrated serine protease of the subtilisin family, previously untested for wound debridement, was incorporated into the emulsion. This device exhibited excellent storage stability. Moisture from the wound triggered an even, reproducible, and complete release of the enzyme within the first 8 hours. The device maintains a moist wound environment that allows the enzyme to achieve nearly complete digestion of the hardened eschar of full‐thickness burns in a porcine model after an exposure period of 24 hours. Debridement was faster than in untreated wounds or wounds treated with a currently available enzyme ointment. Following rapid enzymatic debridement, healing appeared to progress normally, with no histological evidence of damage to adjacent healthy tissue.

Comparative NMR study on the impact of point mutations on protein stability of Pseudomonas mendocina lipase

In this work we compare the dynamics and conformational stability of Pseudomonas mendocina lipase enzyme and its F180P/S205G mutant that shows higher activity and stability for use in washing powders. Our NMR analyses indicate virtually identical structures but reveal remarkable differences in local dynamics, with striking correspondence between experimental data (i.e., 15N relaxation and H/D exchange rates) and data from Molecular Dynamics simulations. While overall the cores of both proteins are very rigid on the pico‐ to nanosecond timescale and are largely protected from H/D exchange, the two point mutations stabilize helices α1, α4, and α5 and locally destabilize the H‐bond network of the β‐sheet (β7–β9). In particular, it emerges that helix α5, undergoing some fast destabilizing motions (on the pico‐ to nanosecond timescale) in wild‐type lipase, is substantially rigidified by the mutation of Phe180 for a proline at its N terminus. This observation could be explained by the release of some penalizing strain, as proline does not require any “N‐capping” hydrogen bond acceptor in the i+3 position. The combined experimental and simulated data thus indicate that reduced molecular flexibility of the F180P/S205G mutant lipase underlies its increased stability, and thus reveals a correlation between microscopic dynamics and macroscopic thermodynamic properties. This could contribute to the observed altered enzyme activity, as may be inferred from recent studies linking enzyme kinetics to their local molecular dynamics.

Enhanced immunogenicity of a functional enzyme by T cell epitope modification

BackgroundT helper epitopes are necessary for the induction of high titers of antigen-specific IgG antibodies. We are interested in the epitope modification of intact proteins as a method to enhance their immunogenicity for the generation of recombinant protein-based vaccines.ResultsHartley strain guinea pig T cell epitopes were mapped for two related bacterial proteases. Two T cell epitopes were found in one of the proteases, while a comparatively reduced immunogenicity protease had no detectable T cell epitopes. A T cell epitope sequence homologous to the immunogenic protease was created in the less immunogenic protease by changing a single amino acid. Proliferative responses to the whole protein parent enzyme were two-fold higher in splenocyte cultures from variant-immunized animals. We found that the single amino acid change in the variant resulted in a protein immunogen that induced higher titers of antigen-specific IgG antibody at low doses and at early time points during the immunization protocol. The serum from parent- and variant-immunized guinea pigs cross-reacted at both the protein and the peptide level. Finally, animals primed to the variant but boosted with the parent enzyme had higher levels of antigen-specific IgG than animals immunized with the parent enzyme alone.ConclusionsWith a single amino acid change we have introduced a T cell epitope into a comparatively low-immunogenic enzyme and have increased its immunogenicity while retaining the enzymes original proteolytic function. The ability to immunomodulate proteins while leaving their function intact has important implication for the development of recombinant vaccines and protein-based therapeutics.

Structural Changes Leading to Increased Enzymatic Activity in an Engineered Variant of Bacillus Lentus Subtilisin

Much of the recent effort of subtilisin protein engineering has centered on the subtilisin from Bacillus lentus. This enzyme has higher alkaline performance than either subtilisin BPN’ from Bacillus amyloliquefaciens or subtilisin Carlsberg from Bacillus licheniformis. While the amino acid sequence of B. lentus subtilisin differs at 106 positions from subtilisin BPN’, including six deleted residues at positions 37a, 58, and 161 to 164, the three-dimensional structures of these subtilisins are very similar and it is possible to draw direct correlations between them.

Inhibitory Complexes of N10- formyltetrahydrofolate Synthetase Indicate Negative Cooperativity between Subunits

N 10 -Formyltetrahydrofolate synthetase (FTHFS) catalyzes the formylation of tetrahydrofolate (H4folate) in an ATP dependent reaction, an initial step in the reduction of carbon dioxide and other one-carbon precursors to acetate (and the reverse reaction). The enzyme, present at high levels in the acetogenic and purinolytic bacteria, is involved in a C1 carbon fixation process for cellular biosynthesis or in ATP synthesis. Here, we report the crystal structures of two nonisomorphous, inhibitory complexes of FTHFS from Morella thermoacetica. The first, obtained at ca 2.0 M ammonium sulfate, is with a catalytic intermediate - formylphosphate, obtained from ATP, and an additional molecule of ATP, which crowds the active site. This explains previously observed substrate inhibition [1]. The other structure is an inhibitory complex of FTHFS with antifolate ZD9331, which crystallized from PEG. Structures have been determined by molecular replacement with the structure of native FTHFS (pdb code: 1EG7) as the starting model and refined using CNS to R factors of 20.4 and 20.1, respectively. The ligands positions allowed us to identify the active site. In both structures the aromatic moieties of ATP and ZD9331 are sandwiched between Phe384 and Trp412. The overlap of the ligands binding sites suggests a double displacement mechanism for FTHFS catalysis rather than the previously proposed random sequential mechanism [2]. FTHFS functions as a tetramer, loose dimer of tightly bound dimers. In both complexes (and in the native structure) the loose interaction corresponds to two-fold crystallographic symmetry. Tight dimmers are asymmetric and there is only one formylphosphate/ATP or ZD9331 per dimmer. Moreover, the complex asymmetry correlates with the crystal packing generating perfectly ordered systems; this is observed for both complexes despite very different crystal packing. The asymmetry of the complexes thus indicates strong negative cooperativity between the tight dimer subunits. An analysis of interactions that communicate ligand binding in subunit A to subunit B and prevent simultaneous ligand binding in subunit B will be presented.