- "Cell-free synthesis and functional characterization of sphingolipid synthases from parasitic Trypanosomatid protozoa"
Sevova ES, Goren MA, Schwartz KJ, et al., Journal of Biological Chemistry 285(27): 20580-20587 July 2010. Results represent the first application of cell-free synthesis for the rapid preparation and functional annotation of integral membrane proteins and thus illustrate its utility in studying otherwise intractable enzyme systems.
- "The length of the bound fatty acid influences the dynamics of the acyl carrier protein and the stability of the Thioester bond"
Zornetzer GA, Tanem J, Fox BG, et al., Biochemistry 49 (3): 470-477 Jan 2010. Acyl carrier proteins involved in fatty acid biosynthesis have been shown to exhibit a high degree of conformational flexibility, in that they are able to sequester fatty acid intermediates between 4 and 18 carbons in length. This flexibility has been observed in X-ray and NMR structures of acyl carrier proteins attached to different fatty acids. NMR studies comparing decanoyl-ACP and stearoyl-ACP indicated that ACP exhibits more dynamic motions when bound to longer fatty acids. We have used complementary chemical and NMR methods as an approach to Improving Our understanding of the effect of fatty acid length on the dynamics of acyl carrier protein
- "Cell-free translation of integral membrane proteins into unilamelar liposomes"
Goren MA, Nozawa A, Makino S, et al., Guide to Protein Purification, Second Edition 466: 647-673 2009. Wheat germ cell-free translation is shown to be an effective method to produce integral membrane proteins in the presence of unilamelar liposomes. In this chapter, we describe the expression vectors, preparation of mRNA, two types of cell-free translation reactions performed in the presence of liposomes, a simple and highly efficient purification of intact proteoliposomes using density gradient ultracentrifugation, and some of the types of characterization studies that are facilitated by this facile preparative approach
- "One-plasmid tunable coexpression for mycobacterial protein-protein interaction studies"
Chang Y, Mead D, Dhodda V, et al., Protein Science 18 (11): 2316-2325 Nov 2009. A single plasmid that allows controlled coexpression has been developed for use in mycobacteria. The tetracycline inducible promoter, PtetO, was used to provide tetracycline-dependent induction of one gene, while the Psmyc, Pimyc, or Phsp promoters were used to provide three different levels of constitutive expression of a second gene.
- "Crystallographic and catalytic studies of the peroxide-shunt reaction in a diiron hydroxylase"
Bailey LJ, Fox BG, Biochemistry 48 (38): 8932-8939 Sep 2009. Here the authors show that toluene 4-monooxygenase has a peroxide-shunt reaction that is similar to 600-fold slower than catalysis driven by biological electron transfer. However, the toluene 4-monooxygenase hydroxylase-effector protein complex was stable in the presence of 300 mM H2O2, suggesting overall benign effects of the exogenous oxidant on active site structure and function.