We are working on enzyme engineering to convert methane to alcohols. As part of this process we wish to measure methane binding to various enzymes and would benefit from suggestions as to what has proved useful to others.
Dear John,
Can isotopic (D or T) methane be used in detection? If yes, it can be passed through a solution of candidate proteins. Subsequent precipitation of the protein solution with methanol or acetone can be followed by isotope detection (unbound methane will not be precipitated). Alternatively, the candidate proteins can be immobilized on plate surfaces, wells of the plate be exposed to methane, and then subjected to isotope detection (I don't know how).
If the methane oxidizing enzyme is iron-dependent, it may involve change in ferromagnetism of the iron moieties. Could that possibly be exploited, somehow, as a detection of methane binding?
If it is agreeable that the methane oxidizing enzyme hydroxylates a methyl group on a larger backbone (say, methyl-lysine), then it will be much easier for detection, because several well-established chemistry for the backbone can be exploited in detection.
I presume you are specifically interested in binding (kon/koff) as opposed to kinetics of reaction?
if your kd are in range of the technique, try investigating isothermal calorimetry (might need to find someone with one that handles gases). Otherwise, more information about the specific enzymes would be needed before more suggestions can be listed e.g. is metal binding involved (if iron what about epr)? Can resonance raman be used? What about comparing ratios or apo and holoenzyme on an orbitrap? Is there a specific Uv-Vis band that can be used for stopped-flow analysis? Is there a large conformational movement that would allow fluorescent quenching to be used? Also, where on the spectrum of detailed to high-throughput would you like the assay to be?
Thanks for these suggestions. High throughput would be great, Ideally we would separately measure methane binding from possible reaction or activation steps.
Working with gasses can be quite complex and usually requires special equipment. The easiest way would be to use something that competes with methane binding. For example, in heme proteins you might measure the competition between O2 and CO, which can be measured spectroscopically and using solutions that are saturated with O2 and CO. None of the methods I can think of off the top of my head are high-throughput as such though when dealing with gasses - mostly because of outgassing from the solvent.
Maybe something using NMR? Deuterated methane would probably overlap with protein peaks so you could perhaps use protein peaks to measure the ratio of bound and unbound enzyme when mixing two buffers, one of which is saturated with methane, and then sealing the NMR tube.
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