OBJECTIVE: I am a protein Biochemist with expertise in difficult protein expression, purification and biophysical characterization. My most recent projects have been on bacterial virulence secretion system APTase enzymes and Novel Antimicrobials for •••••ei and Y. pestis. I am searching for a position where I can continue work on biologics and gain additional experience, potentially leading a project and helping others with my expertise.
PLACE OF BIRTH: Legnica, Poland
MAJOR RESEARCH INTERESTS: Enzymology, Protein-Ligand Interactions, Vaccinology, Protein Stability,
RECENT POSITION: Contractor (•••••year, •••••) Sr. Research Scientist II
LABORATORY ADDRESS: ••••• Blackhawk Rd., Bldg. E•••••, Aberdeen Proving Ground, MD
HOME ADDRESS: •••••, Greencastle, PA •••••
TELEPHONE: ••••• (Cell)
Protein Expression, Purification and Biophysical Characterization
Protein Characterization & Protein-Ligand Interactions
Dec •••••July ••••• Research and Development Command-Edgewood Chemical Biological Center, Sr. Research Scientist (Lab Manager), Aberdeen Proving Ground, MD.
Identification of small molecule inhibitors of bacterial type III secretion system ATPase. The project is a continuation of work started at US Army Medical Research Institute of Infectious Diseases, Ft. Detrick. The target protein was identified using system biology methods and confirmed in an animal model of bacterial infection under BSL••••• conditions. To find the small molecule inhibitors of bacterial ATPases (YscN and BsaS), I expressed the catalytic domain (YscN) and the full length (BsaS) enzymes fused to polyhistidine (C-terminus) and maltose binding protein (N-terminus, cleavable) tags. The proteins were purified under native conditions and the basic kinetic parameters (Km, Vmax, cooperativity) were determined in vitro using phosphate and ADP release assays. The virtual library of small compounds (ZINC, 3D version, 5+ million compounds) was screened computationally for binding to the active site of the enzymes by a collaborator and the best ••••• compounds were purchased and tested in in vitro primary assays in my laboratory. The most promising candidates were selected for in vivo secondary assays in attenuated Y. pestis to determine inhibition of protein secretion by the small molecules in a bacterial cell culture. The biological activity correlated poorly with the in vitro data and analysis of the docking poses of the molecules to the model of recombinant enzymes showed that the molecules most effective in biological assays were most likely preventing assembly of an active hexamer of ATPases in the conformation required for ATP hydrolysis. Detailed screen of the inhibitors showed EC••••• values in the nanomolar range. The best candidates from biological assay were not toxic to multiple mammalian cell lines in a cell culture assay. Based on the combined results from the assays, a new set of small molecules was selected from an extended synthetic organic library database to be custom made by synthetic organic chemistry methods. At present, the molecules are being made and the next round of selection is expected to start in September ••••• The project was in collaboration with scientists from US Army Research Institute of Infectious Diseases, Ft. Detrick and Enamine, a commercial company with expertise in synthetic organic chemistry. At RDECOM, I trained two technicians in cell assays, enzyme kinetics and protein purification.
Dec •••••Dec ••••• U.S. Army Medical Research Insitute of Infectious Diseases, Research Scientist, Ft. Detrick, MD.
Investigation of novel therapeutic targets for blockage of type III secretion system from Y. pestis. The project was designed to analyze the system by system biology methods and identify proteins suitable for target drug development effective against Y. pestsis. The secretory system used by the pathogen is used by many Gram-negative pathogenic bacteria and the components and the function are conserved across different species. I have analyzed the available literature data and the role of different proteins in the secretion process by bioinformatics methods. The analysis suggested that a single ATPase, YscN in Y. pestis, is always present in the system and mutations in the catalytic site of the ATPase blocked the protein secretion by the bacterial system. The protein was designated as a potential therapeutic target for Y. pestis and the gene coding for the protein was deleted using molecular biology methods. The mutated pathogen had virulence attenuated at least million-fold in a mouse model of Y. pestis infection, confirming the target for therapeutics development. The recombinant protein was expressed in E. coli and purified using column chromatography. The ATPase activity of the protein was confirmed in an in vitro assay. The project was transferred in ••••• to Research and Development Command-Edgewood Chemical Biological Center, MD for continuation.
Study of type III secretion system protein-protein interactions. The project was aimed at elucidating the interactions between components of type III secretion system components from Y. pestis and their role in protein transport by the bacteria. I used the recombinant components of the system, most of the secreted proteins and some components of the secretion machinery to measure individual binding by surface plasmon resonance (Biacore). The interactions were classified by the relative strength of the response (relative resonance units) and I prepared the complexes of the best candidates for examination by measurements using MALDI-TOF mass spectroscopy. Most of the interactions were confirmed and the stoichiometry of unusual pairs was confirmed based on the mass assignments of intact complexes. The complexes were also examined by SEC which confirmed stable complexes. I measured their strength (kon, koff, Kd) in detail by surface plasmon resonance. I analyzed the final results in view of the known mechanisms of protein transport by the type III secretion system from Y. pestis and proposed a kinetic order of transport based on all the data. The approach showed a new way to elucidate unknown interactions in a biological system and the results were published in the Journal of Biological Chemistry.
Dec •••••Dec ••••• U.S. Army Medical Research Insitute of Infectious Diseases, Senior Research Fellow, Ft. Detrick, MD.
Elucidation of mechanism of human immune system inactivation by Spe-C superantigen from S. pyogenes. The Spe-C protein is known to stimulate T-cell proliferation by interacting directly with human cells expressing MHC class II and T-cell receptors. The interaction bypasses the specific recognition of protein antigen fragments on MHC class II-expressing cells by T-cell receptors, starting a signaling cascade leading to toxic shock syndrome in humans. To help understand the mechanism of the process, I expressed the recombinant Spe-C in E. coli. The protein was very hydrophobic and had to be refolded on-column to obtain a soluble protein. I used the recombinant protein in mammalian cell culture assays to show stimulation of T-cell receptors and measure its biological potency. The recombinant protein was fully active as compared to the wt protein and had a stable tertiary and secondary structure. To investigate the mechanism in detail, I mutated the residues comprising MHC class II binding site in the recombinant protein and used the mutants to measure binding to cells expressing the MHC II molecules on the surface and their ability to stimulate T-cell proliferation. The mutations showed that there were two binding sites for MHC II molecules: a low affinity site requiring zinc and a higher affinity site important for coupling with T-cell receptors. I confirmed the presence of the first site by Biacore measurements and the second one by T-cell stimulation. Based on the results, I proposed a two-step model of the coupling of MHC II and T-cell receptors by Spe-C. The work was published in the Journal of Biological Chemistry. At USAMRIID I trained many students in Biacore measurements of protein-protein interactions.
Nov •••••Nov ••••• Case Western Reserve University, Postdoctoral Fellow, Cleveland, OH.
Identification of a link between structure and pathogenicity of human prion protein. The abnormal prion protein is thought to be the only infectious agent responsible for human prion diseases. I used a combination of site-directed mutagenesis, stability measurements (CD, fluorescence, SEC), proteinase K digests and thioflavin T assays to characterize multiple variants of the wild type and mutant proteins present in familial prion diseases and identify their aggregation properties and propensity to form the abnormal form of prion protein. I made the isotopically labeled protein for structural studies. The NMR data of the mutant proteins showed no changes in the structure beyond local regions but analysis of the X-ray data showed that the mutants may prevent normal monomer-dimer equilibrium in the wild type protein. I hypothesized that the mutations may be shifting natural equilibrium in the prion protein leading to diminished ability of the human host to remove the mutated protein. The findings were published in Biochemistry, Journal of Biological Chemistry, and Nature Structural Biology.
••••• Lab of Biophysical Chemistry, Post-Doctoral Fellow, Case Western Reserve University. Stability and structure relationship in the human prion protein.
EDUCATION (GRADUATE & POST-DOCTORAL)
••••• Lab of Integrated Toxicology, Post-Doctoral Fellow, US Army Medical Research Institute of Infectious Diseases. Investigation of human immune system inactivation by Spe-C superantigen from S. pyogenes.
••••• Biochemistry, Biochemistry and Molecular Biology (BCMB), Ph. D., University of Gainesville
School of Medicine, Benjamin M. Dunn Laboratory. Enzyme mechanisms and kinetic analysis of a Hepatitis A Virus 3 C protease, a viral protein processing enzyme.
••••• Chemistry with conc. in Physical Chemistry, M. Sc., University of Wroclaw, Poland
UNDERGRADUATE GPA: •••••
SUMMARY OF RELEVANT SKILLS
DNA Cloning, Site-directed Mutagenesis, Gene Knockout in Bacteria
Human and Bacterial Recombinant Protein expression •••••, Yeast and Human Cells)
Protein purification (AKTA FPLC, BioLogic DuoFlo), Column chromatography (AKTA)
Enzyme Kinetics (Pre-steady state & Steady State, high throughput screening, HTS)
Circular Dichroism (Spectra Deconvolution, Tm, Cm)
Fluorescence anisotropy for ligand binding, FRET, TR-FRET
Western blotting and Co-immunoprecipitation, SDS-PAGE
Isotopic Labeling of Proteins, Chemical labeling of proteins (chemicals and fluorophores)
Surface Plasmon Resonance (Biacore, FlexChip)
Ligand and metal binding studies
Computer modeling (homology modeling, ligand binding, oligomer assembly)
HONORS AND DISTINCTIONS
••••• Invited Speaker for Protein Engineering Group, Cambridge, MA
••••• Awarded the National Science Foundation Post-doctoral Fellowship ($•••••,•••••/year for 3 years)
••••• Awarded the Defense Threat Reduction Agency Grant ($1 million/3 years)
••••• Co-PI: In-house ILIR project ($•••••K/year)
••••• Awarded the Defense Threat Reduction Agency Grant ($1 million/2 years)
Secret Security clearance
CDC Registration for working with attenuated Yersinia pestis
STRUCTURES DETERMINED BY NMR AND X-RAY CRYSTALLOGRAPHY
Preparation of proteins
1FO7 NMR Structure of E•••••K mutant prion protein, familial disease
1FKC NMR Structure of E•••••K mutant prion protein, familial disease
1I4M Crystal Structure of human prion protein, wild type
••••• Raab, R. and W. •••••, Yersinia pestis YopD ••••• fragment is partially unfolded in the native state. Protein Expr Purif, ••••• •••••(1): p. •••••
••••• •••••, W., B.S. Powell, and J. Goodin, Yersinia pestis Yop secretion protein F: purification, characterization, and protective efficacy against bubonic plague. Protein Expr Purif, ••••• •••••(1): p. •••••
••••• •••••, W., Barnie, A.M., Dyas, B.K., and Ulrich, R.G., Zinc binding and dimerization of Streptococcus pyogenes pyrogenic exotoxin C are not essential for T-cell stimulation. J Biol Chem, ••••• •••••(•••••): p. •••••
••••• •••••, W., OBrien, S., Holman, K., Cherry, S., Brueggemann, E., Tropea, J.E., Hines, H.B., Waugh, D.S., and Ulrich, R.G., Novel protein-protein interactions of the Yersinia pestis type III secretion system elucidated with a matrix analysis by surface plasmon resonance and mass spectrometry. J Biol Chem, ••••• •••••(•••••): p. •••••
••••• •••••, W., Folding aggregated proteins into functionally active forms. Curr Opin Biotechnol, ••••• •••••(4): p. •••••
••••• Gabus C, Auxilien S, Pchoux C, Dormont D, •••••, W., Morillas M, Surewicz W, Nandi P, Darlix JL., The prion protein has RNA binding and chaperoning properties characteristic of nucleocapsid protein NCP7 of HIV••••• J Biol Chem, ••••• •••••(•••••): p. •••••
••••• Derrington, E., Gabus, C., Leblanc, P. J, Grave, L., Dormont, D., •••••, W., Morillas, M., Marck, D., Nandi, P., Darlix, J.L. .PrPC has nucleic acid chaperoning properties similar to the nucleocapsid protein of HIV••••• C R Biol, ••••• •••••(1): p. •••••
••••• Gabus, C., Auxilien, S., Pchoux, C., Dormont, D., •••••, W., Morillas, M., Surewicz, W., Nandi, P., Darlix, ••••• prion protein has DNA strand transfer properties similar to retroviral nucleocapsid protein. J Mol Biol, ••••• •••••(4): p. •••••
••••• Knaus, K.J., Morillas, M., •••••, W, Malone, M., Surewicz ,W.K., and Yee, V.C. Crystal structure of the human prion protein reveals a mechanism for oligomerization. Nat Struct Biol, ••••• 8(9): p. •••••
••••• Li, R., Liu, T., Wong, B.S., Pan, T., Morillas, M., •••••, W., ORourke, K., Gambetti, P., Surewicz, WK, and Sy, M.S., Identification of an epitope in the C terminus of normal prion protein whose expression is modulated by binding events in the N terminus. J Mol Biol, ••••• •••••(3): p. •••••
••••• Zhang, Y., •••••, W., Zagorski, M.G., Surewicz, W.K., and Snnichsen, F.D., Solution structure of the E•••••K variant of human prion protein. Implications for the mechanism of pathogenesis in familial prion diseases. J Biol Chem, ••••• •••••(•••••): p. •••••
••••• •••••, W., Morillas, M, Chen, S.G., Gambetti, P., and Surewicz, W.K., Aggregation and fibrillization of the recombinant human prion protein huPrP••••• Biochemistry, ••••• •••••(2): p. •••••
••••• Morillas, M., •••••, W., Gambetti, P., and Surewicz, W.K., Membrane environment alters the conformational structure of the recombinant human prion protein. J Biol Chem, ••••• •••••(•••••): p. •••••
••••• •••••, W., Petersen, R.B., Gambetti, P., Surewicz, W.K. Familial mutations and the thermodynamic stability of the recombinant human prion protein. J Biol Chem, ••••• •••••(•••••): p. •••••
••••• •••••, W., Petersen, R., Gambetti, P., and Surewicz, W.K. pH-dependent stability and conformation of the recombinant human prion protein PrP(•••••). J Biol Chem, ••••• •••••(•••••): p. •••••
••••• Johansson, P.J., Malone, C., •••••, W., Dunn, B.M., and Williams, R.C. ••••• structure of monoclonal antibody II••••• against herpes simplex virus Fc gamma-binding glycoprotein gE contains immunodominant complementarity determining region epitopes that react with human immunoglobulin M rheumatoid factors. J Exp Med, ••••• •••••(5): p. •••••
••••• Dunn, B.M., Scarborough, P.E., Davenport, R., and •••••, W. .Analysis of proteinase specificity by studies of peptide substrates. The use of UV and fluorescence spectroscopy to quantitate rates of enzymatic cleavage. Methods Mol Biol, ••••• •••••: p. •••••
••••• Jewell, D.A., ••••• W, Dunn, B.M., Malcolm, B.A., Hepatitis A virus 3C proteinase substrate specificity. Biochemistry, ••••• •••••(•••••): p. •••••
••••• ••••• et al. Identification of small molecule inhibitors of Yersinia pestis YscN ATPase Manuscript in preparation.
(1) James Markarian, (Senior Project Manager), US Army at ECBC, •••••, •••••,
(2) Kamal Saikh, Ph.D., (Coworker), US Army Medical Research Institute of Infectious Diseases, •••••, •••••
(3) Mark Olson, Ph.D. (Collaborator), US Army Medical Research Institute of Infectious Diseases, •••••, •••••