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

Abstract

The transition of the colicin El channel polypeptide from a water-soluble to membrane-bound state occurs in vitro at acid p H 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 p H 3-6 up to 70 "C implies that the colicin E l channel polypeptide is more stable than that of colicin A. A transition between 53 and 58 "C in the amplitude of the near-UV C D is consistent with preservation of part of the hydrophobic core in a destabilized state at p H 2. Thus, the unfolded state associated with colicin activity at acidic p H 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-A globular colicin channel polypeptide in the 10-A 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.

Adventures in Membrane Protein Topology

“…the membrane surface. This suggests that the protein unfolds and spreads out on the surface of the membrane, which is in agreement with the previous proposals for colicin A (26 -28) E1 (29,31,(47)(48)(49) and I a (25,(41)(42)(43). The em max values for W-355 and W-367 single Trp proteins indicate that both helices 1 and 2 are membrane-associated under the conditions used in this study but that the corresponding Trp residues are in a more polar environment than those found within the hydrophobic anchor domain of the channel protein (Table I).…”

“…The em max data for the contingent of single Trp mutant and WT proteins strongly suggest that the polarity of the Trp residues (at 11 of the 14 different Trp sites within the peptide) increases when the protein associates with (51); biotinylation (41,42,53); epitope mapping (43); saturation mutagenesis (55); protease accessibility (28,47); depth-dependent fluorescence quenching (29,31); cysteine-scanning mutagenesis (56); FRET (27); CD, Fourier transform infrared radiation, and differential scanning calorimetry (48,49); time-resolved spin labeling (57); and acrylamide quenching and other fluorescence data presented in this work. The initial surface-bound structure adopts at least two states with state 2 being the most populated.…”

Acid destabilization of the solution conformation of Bcl‐XL does not drive its pH‐dependent insertion into membranes

“…This result is notable because it differs from many proteins with solution conformations and acidic isoelectric points similar to Bcl-x L DTM (calculated pI of 4.4), such as diphtheria toxin translocation domain, colicin A, and colicin B. In these cases, the primary driving force is the acid-induced destabilization of the solution conformation and not simply the acidification of side chains that favors the membrane conformation as we find for Bcl-x L DTM (Ramsay et al 1989;London 1992;Schendel and Cramer 1994;Lesieur et al 1997;Sathish et al 2002;Zakharov and Cramer 2002a)). For example, the rate-limiting step for the solution to membrane conformational change of colicin A is the acidinduced unfolding rate of the solution conformation in the absence of lipid vesicles, and not membrane binding (van der Goot et al 1991).…”

Colicin Biology

“…Interestingly, the maximum channel activity of colicin Ia correlated with conditions that maximized helix rigidity, suggesting that the helices insert into the bilayer as preformed units (688). Further complicating the picture, it has been proposed that colicins A and B interact with the membrane via a molten-globule intermediate in which the secondary structure is retained while the tertiary structure is lost (192,640), whereas this is not the case with colicin E1 (577,692) and colicin N (192). Presumably, the discrepancies among these models represent real differences among the colicins, despite their seductively similar crystal structures.…”