Solution NMR Studies of Antiamoebin, a Membrane Channel-Forming Polypeptide

Abstract

Antiamoebin I is a membrane-active peptaibol produced by fungi of the species Emericellopsis which is capable of forming ion channels in membranes. Previous structure determinations by x-ray crystallography have shown the molecule is mostly helical, with a deep bend in the center of the polypeptide, and that the backbone structure is independent of the solvent used for crystallization. In this study, the solution structure of antiamoebin was determined by NMR spectroscopy in methanol, a solvent from which one of the crystal structures was determined. The ensemble of structures produced exhibit a right-handed helical C terminus and a left-handed helical conformation toward the N-terminus, in contrast to the completely right-handed helices found in the crystal structures. The NMR results also suggest that a "hinge" region exists, which gives flexibility to the polypeptide in the central region, and which could have functional implications for the membrane insertion process. A model for the membrane insertion and assembly process is proposed based on the antiamoebin solution and crystal structures, and is contrasted with the assembly and insertion mechanism proposed for other ion channel-forming polypeptides.

Antiamoebin I in Methanol Solution: Rapid Exchange between Right‐Handed and Left‐Handed 310‐Helical Conformations

“…The stereospecific assignment for H-atoms in prochiral CH 2 groups and for Me groups of Aib residues was achieved by a combined analysis of vicinal J couplings and intensities of ROESY cross-peaks. The assignments for 1 H-and proton-bearing 13 C-nuclei are in agreement with those reported previously by Galbraith et al [28].…”

“…The 13 C constant time HSQC spectrum for the stable isotope-labeled peptide in CD 3 OH solution ( Fig. 1, a) is identical to the HSQC spectrum published by Galbraith et al for Aam-I at natural isotopic abundance [28]. Absence of surplus cross-peaks in this spectrum along with an inspection of the HPLC chromatogram (not shown) indicated purity of the peptide sample.…”

“…508) bend at Hyp 10 [16] [25] [26]. At the same time, it was reported that the N-terminus of Aam-I is folded in a left-handed helix both in DMSO and in MeOH solutions [27] [28].…”

“…This part of the peptide (Table 1) contains five achiral residues (Aib, Gly), two l-residues (Phe, Leu), and one d-residue (Iva). The presence of five a,adialkylated glycyl residues (Aib, Iva) implies a strong helical propensity for this segment, but the helix-handedness in solution remains questionable [27] [28].…”

Solvent Effects on the Secondary Structure of the Membrane‐Active Zervamicin Determined by PELDOR Spectroscopy

“…Since the structure of a peptaibol might be dependent on the environment [7] [8], the Zrv structure was also investigated in various membrane-mimicking solvents (mixtures of CD 3 Cl 3 , CD 3 OH, and H 2 O). The NMR results, however, showed that the conformation appears rather solvent-independent [6] [9].…”