Membrane-Binding Amphipathic .alpha.-Helical Peptide Derived from CTP:Phosphocholine Cytidylyltransferase

Abstract

A peptide corresponding to a portion of the amphipathic alpha-helical region of CTP:phosphocholine cytidylyltransferase was synthesized. This region of the enzyme was proposed to be the membrane-binding domain [Kalmar, G.B., Kay, R.J., Lachance, A., Aebersold, R., & Cornell, R.B. (1990) Proc. Natl. Acad. Sci. U.S.A. 87, 6029]. We have shown that the peptide is physically associated with PG vesicles. CD of the peptide in buffer suggested a primarily random structure, while, in the presence of trifluoroethanol, the peptide was alpha-helical. Anionic lipid vesicles promoted an alpha-helical conformation, whereas neutral or cationic lipid vesicles did not alter the random structure of the peptide, suggesting a selective stabilization of the alpha-helix by anionic membranes. The fluorescence of the single tryptophan residue, which lies on the hydrophobic face of the amphipathic alpha-helix, was studied. Anionic lipid vesicles specifically induced a shift in the fluorescence to a lower wavelength. Fluorescence quenching by the aqueous-phase quencher, I-, and the lipid-phase quencher 9,10-dibromo-PC was used to determine the accessibility of the tryptophan to each of these environments. The presence of anionic lipid vesicles, but not nonanionic lipid vesicles, decreased the quenching by I- suggesting that, in the presence of anionic lipids, the tryptophan residue is poorly accessible to the aqueous I-. Dibromo-PC significantly quenched the fluorescence only when present in anionic vesicles, confirming the membrane location of the tryptophan residue and the lipid specificity of this interaction.(ABSTRACT TRUNCATED AT 250 WORDS)

Molecular dynamics investigation of the influence of anionic and zwitterionic interfaces on antimicrobial peptides’ structure: Implications for peptide toxicity and activity

“…The results from the simulations can be generalized to other non-AMP membrane-active peptide fragments as well. The simulations confirm various scattered experimental observations that peptide secondary structure is preferentially induced in the presence of anionic membranes [3,18,20,29,32]. The starting structures (left) and the final conformations (right) of the three simulations.…”

“…Variants of magainin (charge ≥ +5) which retained helical structure in presence of POPC membranes were found to be highly hemolytic, while those with reduced helical structure were less hemolytic [9]. There are various other instances where the helicity of peptides and membrane protein fragments is conserved in anionic lipids, but not in zwitterionic lipids [3,18,20,29,32]. Often these differences affect the ultimate cellular functions and structural roles of the proteins.…”

Modulation of CTP:phosphocholine cytidylyltransferase by membrane curvature elastic stress

“…Furthermore, inhibition of CCT by using either biochemical (3)(4)(5)(6) or genetic (7) methods triggers programmed cell death. CCT activity is modulated by the association of its amphipathic helical domain with phospholipid bilayers (1,(8)(9)(10). The enzyme binds tightly to PtdCho bilayers containing fatty acids and diacylglycerol (11)(12)(13)(14)(15)(16).…”

Disruption of CCTβ2 Expression Leads to Gonadal Dysfunction

“…A phospholipid sensor domain, or helical domain (residues 256 to 288), is unique to the mammalian proteins and consists of three consecutive 11-residue repeats that form an amphipathic ␣-helix (30). The helical domain regulates the protein's reversible association with biological membranes (2,18,20,30,31) together with its activity (56, 64). The curvature elastic stress hypothesis (4) provides a theoretical framework for understanding both the positive and negative functions of specific membrane lipids in CCT regulation via the helical domain (4,19).…”