Christopher Koo

Chris is a 2nd year IBiS student in the Rosenzweig lab. He received a BS in Biochemistry from UCLA. Chris can be contacted via email here.


Characterizing particulate methane monooxygenase (pMMO) using cell-free protein synthesis

Widespread hydraulic fracturing has increased worldwide supplies of natural gas, an abundant and cheap resource composed primarily of methane. Consequently, there has been renewed interest in using methane-metabolizing bacteria, called methanotrophs, for the bioconversion of methane gas to liquid fuels (Bio-GTL) as well as for its removal from the atmosphere where it is a potent greenhouse gas. As a natural methane sink, methanotrophs are an integral part of the global carbon cycle, using enzymes called methane monooxygenases (MMOs) to convert methane into methanol. MMOs can also degrade halogenated hydrocarbons, pollutants that pose major health concerns, providing a tool for bioremediation.

Progress towards using methanotrophs for fuel production and bioremediation has been hampered by a lack of detailed knowledge of MMO structure and mechanism. In particular, the membrane-bound or particulate MMO (pMMO) used by most methanotrophs is not well understood. Available crystal structures and biochemical data show that it is a α3β3γ3 trimer containing a copper active site, but low resolution precludes definitive conclusions about the atomic structure of the active site. Despite extensive efforts, recombinant expression of pMMO has not been successful. Mutants can be generated within the native methanotrophs, but thus far, mutagenesis of the active site metal binding residues has been lethal in vivo, preventing further study of these variants. To overcome this cell viability issue, I use cell-free protein synthesis (CFPS) to investigate unaddressed questions about pMMO structure and function.

CFPS uses lysates from cell culture depleted of DNA, mRNA, and cell debris as the source of translation machinery including ribosomes, polymerases and tRNAs. By extracting this machinery, CFPS offers the advantage of an open reaction with precise control over the reactants and permits the expression of toxic proteins. Combined with membrane mimetics such as nanodiscs and bicelles, this system provides an alternative approach to studying pMMO mutants and may provide groundwork for studying other large membrane protein complexes as well.

Recent Photos

September 12, 2016