ON THE NATURE OF THE UNFOLDED INTERMEDIATE IN THE IN-VITRO TRANSITION OF THE COLICIN E1 CHANNEL DOMAIN FROM THE AQUEOUS TO THE MEMBRANE PHASE

The transition of the colicin E1 channel polypeptide from a water-soluble to membrane-bound state occurs in vitro at acid pH values that are associated with an unfolded channel structure whose properties qualitatively resemble those of a ''molten globule,'' or ''compact unfolded,'' intermediate state. The role of such a state for activity was tested by comparing the pH dependence of channel-induced solute efflux and the amplitude of the near-UV CD spectrum. The requirement of a partly unfolded state for activity was shown by the coincidence of the onset of channel activity measured for 4 different lipid compositions with the decrease in near-UV CD amplitude as a function of pH. Tertiary constraints on the 3 tryptophans of the colicin channel, assayed by the amplitude of the near-UV CD spectrum, are retained over the pH range 3-4 where channel activity could be measured and, as well, at pH 2. In addition, the tryptophan fluorescence emission spectrum is virtually unchanged over the pH range 2-6. The temperature independence of the near UV spectrum at pH 3-6 up to 70 degrees C implies that the colicin E1 channel polypeptide is more stable than that of colicin A. A transition between 53 and 58 degrees C in the amplitude of the near-UV CD is consistent with preservation of part of the hydrophobic core in a destabilized state at pH 2. Thus, the unfolded state associated with colicin activity at acidic pH has the properties of a ''compact unfolded'' state, having some, but not all of the properties of a ''molten globule.'' The small effect on local membrane acidity of a physiological acidic membrane lipid content, the retention of significant near-UV CD amplitude down to pH 2, and the small extent of immersion of the 40-Angstrom globular colicin channel polypeptide in the 10-Angstrom lower pH layer at the membrane surface make it unlikely that a local lower pH at the membrane surface significantly facilitates formation of an unfolded intermediate.