EPR OF NO HEMOPROTEINS

Colicin Ia is a 626 amino acid bacterial protein which kills cells by disrupting active transport and by inducing the leakage of ions. It has been shown that this activity derives from the ability of Colicin Ia to form voltage-dependent, relatively non-selective ion-permeable channels in the bacterial plasma membrane. The efficacy of Colicin Ia is remarkable, in that a single protein molecule in the membrane is enough to kill the cell. In addition, the voltage dependent channel forming behavior can be reproduced in artificial phospholipid bilayer membranes and vesicles, thus providing an ideal system for the general study of ion channel physics. High frequencey EPR studies of spin-labeled colicin Ia molecules imbedded in vesicles is an ideal means to address structural details of voltage-dependent ion channel structure. An overview of the proposed experimental protocol is as follows. A paramagnetic nitroxide spin label is introduced into the colicin protein at a residue position of interest. The colicin is then imbedded in membrane vesicles followed by rapid mixing with the depsipeptide valinomycin to establish a transmembrane potential. At this point the system can be rapidly frozen to lock the protein conformational state in place followed by low temperature EPR spectroscopy. Alternatively, solution phase EPR spectra may be obtained. In either case, the EPR spectrum of the nitroxide spin label is influenced by the polarity of the environment, and thus provides a means to establish if the label is outside or within (and perhaps how deep within) the vesicle bilayer. This in turn leads to information concerning the voltage-dependent structure of the protein and how it forms the ion channel.