A Designed Zn²⁺-Binding Amphiphilic Polypeptide:  Energetic Consequences of π-Helicity

Abstract

The pi-helix is a secondary structure with 4.4 amino acids per helical turn. Although it was proposed in 1952, no experimental support for its existence was obtained until the mid-1980s. While short peptides are unlikely to assume a marginally stable secondary structure spontaneously, they might do so in the presence of appropriate structural constraints. In this paper, we describe a peptide that is designed to assume a pi-helical conformation when stabilized by cetyltrimethylammonium bromide (CTAB) micelles and Zn(2+). In the designed peptide, lipophilic amino acids are placed such that it would be amphiphilic in the pi-helical, but not in the alpha-helical, conformation. Also, two His residues are incorporated with i, i + 5 spacing, designed to allow binding of Zn(2+) in a pi-helical but not an alpha-helical conformation. The peptide was found to form moderately stable monolayers at the air-water interface, with a collapse pressure that almost doubled when there was Zn(2+) in the subphase. Also, CTAB micelles induced a marked increase in the helicity of the peptide. In 50% TFE, the peptide had a CD spectrum consistent with an alpha-helical structure. The addition of 1 mM Zn(2+) to this solvent caused a saturable decline in ellipticity to approximately half of its original value. The peptide also bound Zn(2+) when it was bound to CTAB micelles, with Zn(2+) again inducing a decrease in ellipticity. The peptide had slightly greater affinity for Zn(2+) in the presence of the CTAB than in a 50% TFE solution (K(d) = 3.1 x 10(-4) M in CTAB and 2.3 x 10(-4) M in TFE). van't Hoff analysis indicated that thermal denaturation of the peptide in 50% TFE containing 1 mM Zn(2+) was associated with both enthalpic and entropic changes that were greater than those in the absence of Zn(2+). These observations are all consistent with the proposal that the peptide assumed a pi-helical conformation in the presence of Zn(2+) and CTAB micelles, and has allowed the stability of this rare conformation to be assessed.

Molecular Dynamics Simulations of the E1/E2 Transmembrane Domain of the Semliki Forest Virus

“…Similar behavior is exhibited in other MD studies in vacuum and in solvated lipid bilayer environment (Duneau et al, 1999(Duneau et al, , 1996Lee et al, 2000). p-Helical segments, sometimes called a-aneurisms, have been found experimentally (Keefe et al, 1993;Morgan et al, 2001;Rajashankar and Ramakumar, 1996), and they are believed to be intimately involved in protein function (Weaver, 2000). In the case of TM helices, p-helical turns seem necessary to accommodate long residue strings inside the lipid bilayer (Popot and Engelman, 2000).…”

Empirical force fields for biological macromolecules: Overview and issues

“…Verification of this was performed by applying the CMAP approach where, by reproducing the to ␣-helical QM energy difference in a modified CHARMM22 force field, sampling of the -helical conformation was significantly diminished. Although these results do not eliminate the possibility of -helical conformations in proteins, 178 they emphasize the influence a force field can have on the results of MD simulations, and that when novel results are obtained from empirical force field studies, underlying influences associated with the force field being applied should be considered.…”

Force Field Influence on the Observation of π-Helical Protein Structures in Molecular Dynamics Simulations

“…21 However, evidence for π-helicity was observed with CD in a Zn 2+binding amphiphilic peptide that was specifically designed to form a π-helical structure. 10 Further experimental data are available from high-resolution ion mobility measurements of unsolvated and partially hydrated peptides. 28,29 In these studies, collision cross-sections were calculated for simulated peptide conformations and compared with measured values.…”

“…In most of these cases, specific binding interactions have been implicated as stabilizing factors . Another example of how a π-helical conformation may be stabilized was given recently in a designed peptide where two histidines, five residues apart, were aligned to form a zinc-binding site by forming a π-helix . These experimental observations suggest that π-helical structures are only stable under exceptional circumstances.…”