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1. Modular design and self-assembly of multidomain peptides towards cytocompatible supramolecular cell penetrating nanofibers

   https://doi.org/10.1039/D0RA04748A  

   Yang, Su; Dong, He (August 2020, RSC Advances)

   null (Ed.)

   The discovery of cell penetrating peptides (CPPs) with unique membrane activity has inspired the design and synthesis of a variety of cell penetrating macromolecules, which offer tremendous opportunity and promise for intracellular delivery of a variety of imaging probes and therapeutics. While cell penetrating macromolecules can be designed and synthesized to have equivalent or even superior cell penetrating activity compared with natural CPPs, most of them suffer from moderate to severe cytotoxicity. Inspired by recent advances in peptide self-assembly and cell penetrating macromolecules, in this work, we demonstrated a new class of peptide assemblies with intrinsic cell penetrating activity and excellent cytocompatibility. Supramolecular assemblies were formed through the self-assembly of de novo designed multidomain peptides (MDPs) with a general sequence of K x (QW) 6 E y in which the numbers of lysine and glutamic acid can be varied to control supramolecular assembly, morphology and cell penetrating activity. Both supramolecular spherical particles and nanofibers exhibit much higher cell penetrating activity than monomeric MDPs while supramolecular nanofibers were found to further enhance the cell penetrating activity of MDPs. In vitro cell uptake results suggested that the supramolecular cell penetrating nanofibers undergo macropinocytosis-mediated internalization and they are capable of escaping from the lysosome to reach the cytoplasm, which highlights their great potential as highly effective intracellular therapeutic delivery vehicles and imaging probes. 

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2. Insights into Membrane Damage by α-Helical and β-Sheet Peptides

   https://doi.org/10.3390/biom15070973  

   Rangubpit, Warin; Distaffen, Hannah E; Nilsson, Bradley L; Dias, Cristiano L (July 2025, Biomolecules)

   Peptide-induced disruption of lipid membranes is central to both amyloid diseases and the activity of antimicrobial peptides. Here, we combine all-atom molecular dynamics simulations with biophysical experiments to investigate how four amphipathic peptides interact with lipid bilayers. All peptides adsorb on the membrane surface. Peptide M01 [Ac-(FKFE)2-NH2] self-assembles into β-sheet nanofibrils that span both leaflets of the membrane, creating water-permeable channels. The other three peptides adopt α-helical structures at the water–lipid interface. Peptide M02 [Ac-FFKKFFEE-NH2], a sequence isomer of M01, does not form β-sheet aggregates and is too short to span the bilayer, resulting in no observable water permeation across the membrane. Peptides M03 and M04 are α-helical isomers long enough to span the bilayer, with a polar face that allows the penetration of water deep inside the membrane. For the M03 peptide [Ac-(FFKKFFEE)2-NH2], insertion into the bilayer starts with the nonpolar N-terminal amino acids penetrating the hydrophobic core of the bilayer, while electrostatic interactions hold negative residues at the C-terminus on the membrane surface. The M04 peptide, [Ac-FFKKFFEEFKKFFEEF-NH2], is made by relocating a single nonpolar residue from the central region of M03 to the C-terminus. This nonpolar residue becomes unfavorably exposed to the solvent upon insertion of the N-terminal region of the peptide into the membrane. Consequently, higher concentrations of M04 peptides are required to induce water permeation compared to M03. Overall, our comparative analysis reveals how subtle rearrangements of polar and nonpolar residues modulate peptide-induced water permeation. This provides mechanistic insights relevant to amyloid pathology and antimicrobial peptide design. 

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3. Tertiary Plasticity Drives the Efficiency of Enterocin 7B Interactions with Lipid Membranes

   https://doi.org/10.1021/acs.jpcb.3c08199  

   Zhuang, Yi; Quirk, Stephen; Stover, Erica R; Bureau, Hailey R; Allen, Caley R; Hernandez, Rigoberto (March 2024, The Journal of Physical Chemistry B)

   The ability of antimicrobial peptides to efficiently kill their bacterial targets depends on the efficiency of their binding to the microbial membrane. In the case of enterocins, there is a three-part interaction: initial binding, unpacking of helices on the membrane surface, and permeation into the lipid bilayer. Helical unpacking is driven by the dis- ruption of the peptide hydrophobic core when in contact with the membrane. Enterocin 7B is a leaderless enterocin antimicrobial peptide produced from Enterococcus faecalis that functions alone, or with its cognate partner enterocin 7A, to efficiently kill a wide variety of gram-stain positive bacteria. To better characterize the role that tertiary structural plasticity plays in the ability of enterocin 7B to interact with membranes, a series of arginine single site mutants were constructed that destabilize the hydropho- bic core to varying degrees. A series of experimental measures of structure, stability, and function, including CD spectra, far UV CD melting profiles, minimal inhibitory concentrations (MIC) analysis, and release kinetics of calcein, show that decreased sta- bilization of the hydrophobic core is correlated with increased efficiency of a peptide to permeate membranes, and in killing bacteria. Finally, using the computational tech- nique of adaptive steered molecular dynamics, we found that the atomistic/energetic landscape of peptide mechanical unfolding leads to free energy differences between the wild type and its mutants whose trends correlate well with experiment. 

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4. Lung SPLUNC1 Peptide Derivatives in the Lipid Membrane Headgroup Kill Gram-Negative Planktonic and Biofilm Bacteria

   Jakkampudi, Tanvi; Lin, Qiao; Mitra, Saheli; Qin, Weiheng; Kang, Ann; Chen, Jespar; Ryan, Emma; Wang, Runwuan; Gong, Yuqi; Heinrich, Frank; et al (May 2023, Biomacromolecules)

   Lu, Hua (Ed.)

   SPLUNC1 (short palate lung and nasal epithelial clone 1) is a multifunctional host defense protein found in human respiratory tract with antimicrobial properties. In this work we compare the biological activities of four SPLUNC1 antimicrobial peptide (AMP) derivatives using paired clinical isolates of the Gram-negative (G(-)) bacteria Klebsiella pneumoniae, obtained from eleven patients with/without colistin resistance. Secondary structural studies were carried out to study interactions between the AMPs and lipid model membranes (LMMs) utilizing circular dichroism (CD). Two peptides were further characterized using x-ray diffuse scattering (XDS) and neutron reflectivity (NR). A4-153 displayed superior antibacterial activity in both G(-) planktonic cultures and biofilms. NR and XDS revealed that A4-153 (highest activity) is located primarily in membrane headgroups, while A4-198 (lowest activity) is located in hydrophobic region. CD revealed that A4-153 is helical while A4-198 has little helical character, demonstrating that helicity and efficacy are correlated in these SPLUNC1 AMPs. 

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5. Fluorescence cross-correlation spectroscopy of lipid-peptide interactions on supported lipid bilayers

   https://doi.org/bs.abl.2019.01.005  

   Xiaosi Li, Xiaojun Shi (March 2019, Advances in biomembranes and lipid self-assembly)

   Electrostatic interactions drive molecular assembly and organization in the plasma membrane. Specific protein-lipid interactions, however, are difficult to resolve. Here we report on a unique approach to investigate these interactions with time-resolved fluorescence spectroscopy. The experiments were performed on a model membrane system consisting of a supported lipid bilayer with an asymmetric distribution of PIP2 in the upper leaflet of the bilayer. The bilayer also contained nickel-chelating lipids that bind to a histidine-tagged peptide of interest. Both the peptide and the lipid were labeled with orthogonal fluorescent probes, so that diffusion and binding could be measured with two-color, pulsed-interleaved excitation fluorescence cross-correlation spectroscopy (PIE-FCCS). Our PIE-FCCS data showed significant lipid-peptide cross-correlation between PIP2 lipids and membrane-bound cationic peptides. Cross-correlation is a direct indication of lipid-peptide binding and complexation. Together with mobility data, we quantified the degree of binding, which offers new insight into this class of lipid-peptide interactions. Overall, this is the first report of lipid-peptide cross-correlation by FCCS, and provides a new route to quantifying the interactions between proteins and lipid membranes, a key interface in cell signaling. 

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