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MALKOWSKI LABORATORY home > about > Malkowski
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Dr. Michael G. Malkowski is a Senior Research Scientist at the Hauptman-Woodward Medical Research Institute (HWI), an independent, not-for-profit, biomedical research facility located in the heart of downtown Buffalo's medical campus. HWI is the home of Dr. Malkowski’s research laboratories, the High-Throughput Crystallization Screening Laboratory (HTS Lab), and the team responsible for MPSBC's information management systems.

Structural studies of TMP targets in the Malkowski laboratory include:

group1) TP0453. TP0453 is a pore-forming outer membrane-associated lipoprotein of Treponema pallidum that possesses unusual biophysical properties compared to other spirochetal lipoproteins in that it has amphipathic helices that form pores in artificial membranes [1]. The protein was expressed in E. coli, followed by solubilization and purification in the presence of β-octyl glucoside (β-OG). Initial crystallization conditions were identified utilizing a tailored grid screen optimized for TMPs based on detergent phase partitioning [2]. Crystals diffract to better than 2.4Å, and the structure was solved using MAD phasing techniques. The secondary structure of TP0453 consists of twelve β-strands and nine α-helices that generate a unique tertiary fold similar to a α-β-α three-layer sandwich fold.

2) Cyclooxygenases (COX-1 and COX-2). The Cyclooxygenases (COX) are membrane-bound, bifunctional enzymes that catalyze the conversion of arachidonic acid (AA) to Prostaglandin H2 in the committed step of prostaglandin (PG) biosynthesis. The Malkowski laboratory has developed comprehensive protocols for insect cell expression, purification and generation of Co3+-protoporphyrin IX reconstituted COX-2. Reconstitution with Co3+-protoporphyrin IX renders the enzymes catalytically inert and allows for a variety of substrates to be bound within the cyclooxygenase channel [3]. Utilizing these protocols, the Malkowski lab has determined the structures of COX-1 [4, 5, 6] and COX-2 [7] in complex with AA and other polyunsaturated fatty acids, as well as structures of COX-2 with aspirin and endocannabinoid substrates.

3) Pathogen-inducible Oxygenase (PIOX). PIOX is a heme-containing, membrane-associated protein found in mono- and dicotyledonous plants that utilizes stereoselective oxygenation to convert polyunsaturated fatty acids into their corresponding 2R-hydroperoxides [8]. The Malkowski lab has grown crystals of Oryza sativa PIOX Arabidopsis thaliana PIOX utilizing the tailored TMP grid screen [2]. Crystals of O. sativa PIOX and A. thaliana PIOX diffract to greater than 2.2Å using synchrotron radiation.

References

1. Hazlett, K.R., D.L. Cox, M. Decaffmeyer, M.P. Bennett, D.C. Desrosiers, C.J. La Vake, M.E. La Vake, K.W. Bourell, E.J. Robinson, R. Brasseur, and J.D. Radolf, TP0453, a concealed outer membrane protein of Treponema pallidum, enhances membrane permeability. J Bacteriol, 2005. 187(18): p. 6499-508.  (PMID: 16159783)
2. Koszelak-Rosenblum, M., A. Krol, N. Mozumdar, K. Wunsch, A. Ferin, E. Cook, C.K. Veatch, R. Nagel, J.R. Luft, G.T. Detitta, and M.G. Malkowski, Determination and application of empirically derived detergent phase boundaries to effectively crystallize membrane proteins. Protein Sci, 2009. 18(9): p. 1828-1839.  (PMID: 19554626)
3. Malkowski, M.G., M.J. Theisen, A. Scharmen, and R.M. Garavito, The formation of stable fatty acid substrate complexes in prostaglandin H(2) synthase-1. Arch Biochem Biophys, 2000. 380(1): p. 39-45.  (PMID: 10900130)
4. Malkowski, M.G., S.L. Ginell, W.L. Smith, and R.M. Garavito, The productive conformation of arachidonic acid bound to prostaglandin synthase. Science, 2000. 289(5486): p. 1933-7.  (PMID: 10988074)
5. Malkowski, M.G., E.D. Thuresson, K.M. Lakkides, C.J. Rieke, R. Micielli, W.L. Smith, and R.M. Garavito, Structure of eicosapentaenoic and linoleic acids in the cyclooxygenase site of prostaglandin endoperoxide H synthase-1. J Biol Chem, 2001. 276(40): p. 37547-55.  (PMID: 11477109)
6. Thuresson, E.D., M.G. Malkowski, K.M. Lakkides, C.J. Rieke, A.M. Mulichak, S.L. Ginell, R.M. Garavito, and W.L. Smith, Mutational and X-ray crystallographic analysis of the interaction of dihomo-gamma -linolenic acid with prostaglandin endoperoxide H synthases. J Biol Chem, 2001. 276(13): p. 10358-65.  (PMID: 11121413)
7. A.J. Vecchio, D.M. Simmons, and Malkowski, M.G., Structural basis of fatty acid substrate binding to cyclooxygenase-2. J Biol Chem, 2010. 285(29): p. 22152-63. (PMID: 20463020)
8. Koszelak-Rosenblum, M., A.C. Krol, D.M. Simmons, C.C. Goulah, L. Wroblewski, and M.G. Malkowski, His-311 and Arg-559 are key residues involved in fatty acid oxygenation in pathogen-inducible oxygenase. J Biol Chem, 2008. 283(36): p. 24962-71. (PMID: 18596034)
9. Lloyd, T., A. Krol, D. Campanaro, and M.G. Malkowski, Purification, crystallization and preliminary X-ray diffraction analysis of pathogen-inducible oxygenase (PIOX) from Oryza sativa. Acta Cryst, 2006. F62: p. 365-367.  (URL)

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