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

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Abstract
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°C implies that the colicin E1 channel polypeptide is more stable than that of colicin A. A transition between 53 and 58°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-Å globular colicin channel polypeptide in the 10-Å 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.