Membrane Active Peptides and Their Biophysical Characterization

Abstract

In the last 20 years, an increasing number of studies have been reported on membrane active peptides. These peptides exert their biological activity by interacting with the cell membrane, either to disrupt it and lead to cell lysis or to translocate through it to deliver cargos into the cell and reach their target. Membrane active peptides are attractive alternatives to currently used pharmaceuticals and the number of antimicrobial peptides (AMPs) and peptides designed for drug and gene delivery in the drug pipeline is increasing. Here, we focus on two most prominent classes of membrane active peptides; AMPs and cell-penetrating peptides (CPPs). Antimicrobial peptides are a group of membrane active peptides that disrupt the membrane integrity or inhibit the cellular functions of bacteria, virus, and fungi. Cell penetrating peptides are another group of membrane active peptides that mainly function as cargo-carriers even though they may also show antimicrobial activity. Biophysical techniques shed light on peptide⁻membrane interactions at higher resolution due to the advances in optics, image processing, and computational resources. Structural investigation of membrane active peptides in the presence of the membrane provides important clues on the effect of the membrane environment on peptide conformations. Live imaging techniques allow examination of peptide action at a single cell or single molecule level. In addition to these experimental biophysical techniques, molecular dynamics simulations provide clues on the peptide⁻lipid interactions and dynamics of the cell entry process at atomic detail. In this review, we summarize the recent advances in experimental and computational investigation of membrane active peptides with particular emphasis on two amphipathic membrane active peptides, the AMP melittin and the CPP pVEC.

Keywords: antimicrobial peptides; biophysical characterization; cell-penetrating peptides; membrane disruption; peptide–lipid interactions; uptake mechanism.

Figure 1

Number of publications in the past 40 years that use the phrases antimicrobial peptide, cell-penetrating peptide, or membrane active peptide.

Figure 2

Examples of α-helical antimicrobial peptides (AMPs) (top panel) and cell-penetrating peptides (CPPs) (bottom panel). Top panel shows the X-ray crystal structure of melittin (2mlt), solution structure of LL-37 (cathelicidin) in deuterated sodium dodecyl sulfate (dSDS) micelles (2k6o) [34], and the solution structure cecropin A(1-8)–magainin 2(1-12) hybrid peptides (1d9j) [35]. The solution structures of CPPs penetratin in negatively charged phospholipid bicelles (1omq) [36] and transportan in neutral phospholipid bicelles (1smz) [37] are shown in the bottom panel.

Figure 3

Examples of cyclic peptides with a β-sheet core. (A) Solution structure of human defensin 5 (2lxz) [27], (B) crystal structures of crotamine (4gv5) [38], (C) and human β-defensin-2 (1fd3) [20]. Disulfide bridges holding the β-strands together are shown in yellow stick representation.

Figure 4

Different pore models. (A) Shai–Huang–Matsazuki (SHM) model [9]. (B) Electroporation model (reprinted from a previous paper [33], Copyright (2006), with permission from Elsevier). (C) Double-belt pore model (reprinted with permission from a previous paper [41]. Copyright (2014) American Chemical Society).

Figure 5

Major approaches used in biophysical characterization of membrane active peptides. X-ray S/D: X-ray scattering/diffraction; Neutron S/D: Neutron scattering/diffraction; NMR: Nuclear magnetic resonance spectroscopy; CD: Circular dichroism spectroscopy; ITC: Isothermal titration calorimetry; DSC: Differential scanning calorimetry; AFM: Atomic force microscopy; FTIR: Fourier transform infrared spectroscopy; DLS: Dynamic light scattering; CG: Coarse grained models; ESS: Enhanced sampling simulations (SMD-US): (Steered molecular dynamics simulations-Umbrella sampling); MDS: Molecular Dynamics simulations; MC: Monte Carlo calculations; FC: Flow cytometry; FS: Fluorescence spectroscopy; FM: Fluorescence microscopy; Cryo-EM: Cryoelectron microscopy; SPR: Surface plasmon resonance.

Figure 6

Scanning electron microscopy (SEM) imaging of antibiotic resistant and wild-type Escherichia coli (E. coli) cells on membrane filters. Antibiotic resistant E. coli cells in absence of any drugs (A), treated with minimum inhibitory concentration (MIC) of melittin (C), wild-type E. coli cells in absence of any drugs (B), treated with MIC of melittin (D) (reproduced with permission from a previous paper [336]).

Figure 7

SEM imaging of Escherichia coli cells on membrane filters. Untreated E. coli cells (top), E. coli treated with MIC of pVEC (middle), and with 500 μM del5 pVEC peptide (bottom). Changes in surface structure due to peptide treatment are indicated by arrows (Reproduced with permission of the authors of a previous paper [279]).

Figure 8

Translocation of pVEC across the lipid bilayer examined using SMD simulations [304].