O 2 activation in biological systems is a key step in respiration with O 2 activation achieved by a complex series of steps involving binding to an Fe ± heme, electron transfer, and, ultimately, atom transfer to a reducing substrate. Kinetic difficulties in the electroreduction of O 2 to H 2 O in fuel cells create a significant over-voltage which limits
The bacterial small-molecule three-hybrid system described herein should provide a robust platform for high-throughput assays based on protein ± small molecule interactions. The Mtx ± SLF heterodimeric ligand can be prepared readily and gives a strong transcription readout in the E. coli RNA polymerase three-hybrid system. Notably, the levels of transcriptional activation with the Mtx ± SLF three-hybrid system are comparable to those with the direct protein ± protein interaction, despite the fact that one noncovalent interaction has been replaced with two. The EC 50 for lacZ transcription is greater than the K D of either Mtx or SLF for FKBP12. [5] Currently we are carrying out in vitro experiments to examine the relationship between lacZ transcription and the K D of the ligand ± receptor interaction. Three-hybrid systems provide an in vivo alternative to affinity chromatography that can be used to evolve proteins that recognize a particular small molecule, to screen a library of small molecules based on binding to a particular protein, or to screen cDNA libraries to find the protein targets of drugs or to classify proteins based on their small-molecule interactions. Because of the high transformation efficiency and rapid doubling time of E. coli, this system should increase the number of proteins that can be tested in three-hybrid assays by several orders of magnitude compared with yeast systems. A bacterial assay should be particularly advantageous in molecular evolution experiments in which in the order of 10 8 variants may be necessary to alter protein function. Based on our results, we believe that Mtx will provide a versatile anchor for presenting a variety of different small molecules.