Membrane insertion of the pore‐forming domain of colicin A

Abstract

In order to gain some insight into the mechanism of insertion into membranes of the pore-forming domain of colicin A and the structure of its membrane-bound form, circular dichroism (in the near and far ultraviolet), fluorescence and ultraviolet spectroscopy experiments were carried out. Because the structure of the water-soluble form of this fragment has been determined by X-ray crystallography, these spectroscopic methods provided valuable information on the secondary structure and the environment of aromatic residues within the two forms of the peptide. These results strongly suggest that the pore-forming domain of colicin A does not undergo drastic unfolding upon insertion into membrane. The conformational change associated with this process is triggered by the negatively charged lipids and probably consists of a reorientation of helix pairs with respect to each other. Exposure of the aromatic residues to the aqueous phase decreases on binding to lipids whilst the exposure of the tryptophans to the membrane phase increases. This cannot occur without a reorientation of helices 3 -10. All data from this study support the model presented previously in which the known crystal structure opens like an 'umbrella' inserting the hydrophobic hairpin (helix 8 -9) perpendicular to the membrane plane and the helical pair 1-2 and the domain containing the three tryptophans (helices 3-7) lying more or less parallel to the membrane plane. Lipids are bound more tightly to the protein at acidic pH than at neutral pH although a similar lipid protein complex is formed with 1,2-dimyristoyl-sn-glycero(3)-phospho(l)-sn-glycerol at both pH values.Colicins are plasmid-encoded protein antibiotics which kill bacteria closely related to the producing strain (generally Escherichia coli). Like many toxins, colicins are composed of structural domains specialized in one of the different steps of the activity i.e. targeting, translocation and killing. The major group of colicins is the E l class, comprised of colicins A, B, El,

The N-Terminal Domain of Bcl-xL Reversibly Binds Membranes in a pH-Dependent Manner

“…Structurally, Bcl-x L ΔTM is most similar to the translocation domain of diphtheria toxin (23). For this toxin, as well as others, membrane association is mediated in part by the so-called hydrophobic, helical hairpin, which corresponds to α-helices 5 and 6 in Bcl-x L ΔTM (Figure 5a) (38)(39)(40)(41)(42)(43)(44)(45)(46). Despite the structural similarity, the sequences of these proteins are less than 19% identical, and they possibly are an example of convergent evolution toward a versatile protein fold.…”

Interacion of the colicin‐A pore‐forming domain with negatively charged phospholipds

“…A re-orientation of the secondary-structure elements of the protein seems to take place as indicated by fluorescence energy-transfer experiments (Lakey et al, 1991 b). Other changes which occur after membrane insertion have been revealed by near-ultraviolet CD (Lakey et al, 1991a) and differential scanning calorimetry (Muga et al, 1993).A recent approach applied to the determination of membrane-protein topology is the use of brominated phospholipids (Leto et al, 1980;East and Lee, 1982;Markello et al, 1985). Bromine atoms can be easily incorporated into the unsaturated fatty-acid alkyl chain of phospholipids, and the characteristics of the resulting phospholipid are similar to those of the unsaturated precursor (East and Lee, 1982).…”

“…Based on the three-dimensional structure of this fragment, a model for the insertion has been proposed , in which the compact soluble structure, upon interaction with the phospholipid bilayer, un- folds in an umbrella-like fashion. This is the so-called 'umbrella model'.The study of the membrane-bound conformation of colicin A by spectroscopic techniques shows that there is no drastic conformational change upon membrane insertion : the secondary structure of the protein and its fluorescence parameters are largely conserved (Lakey et al, 1991a; Goormagtigh et al, 1992). A re-orientation of the secondary-structure elements of the protein seems to take place as indicated by fluorescence energy-transfer experiments (Lakey et al, 1991 b).…”

“…The study of the membrane-bound conformation of colicin A by spectroscopic techniques shows that there is no drastic conformational change upon membrane insertion : the secondary structure of the protein and its fluorescence parameters are largely conserved (Lakey et al, 1991a;Goormagtigh et al, 1992). A re-orientation of the secondary-structure elements of the protein seems to take place as indicated by fluorescence energy-transfer experiments (Lakey et al, 1991 b).…”

Secondary structure of the membrane‐bound form of the pore‐forming domain of colicin A

“…A previous CD analysis [38] indicated about 53% %-helix at pH 5. However, re-evaluation of the molar absorption coefficient of the peptide would yield a figure close to that from lhc X-ray data.…”