Supramolecular self assembly of nanodrill-like structures for intracellular delivery.
ABSTRACT: Despite recent advances in the supramolecular assembly of cell-penetrating peptide (CPP) nanostructures, the tuning of size, shape, morphology and packaging of drugs in these materials still remain unexplored. Herein, through sequential ligation of peptide building blocks, we create cell-penetrating self-assembling peptide nanomaterials (CSPNs) with the capability to translocate inside cells. We devised a triblock array of Tat48-59 [HIV-1 derived transactivator of transcription48-59] based CPPs, conjugated to up to four Phenylalanine (Phe) residues through an amphiphilic linker, (RADA)2. We observed that the sequential addition of Phe leads to the transition of CSPN secondary structures from a random coil, to a distorted ?-helix, a ?-sheet, or a pure ?-helix. This transition occurs due to formation of a heptad by virtue of even number of Phe. Atomic force microscopy revealed that CSPNs form distinct shapes reminiscent of a "drill-bit". CSPNs containing two, three or four Phe, self-assemble into "nanodrill-like structures" with a coarse-twisted, non-twisted or fine-twisted morphology, respectively. These nanodrills had a high capacity to encapsulate hydrophobic guest molecules. In particular, the coarse-twisted nanodrills demonstrate higher internalization and are able to deliver rapamycin, a hydrophobic small molecule that induced autophagy and are capable of in vivo delivery. Molecular dynamics studies provide microscopic insights into the structure of the nanodrills that can contribute to its morphology and ability to interact with cellular membrane. CSPNs represent a new modular drug delivery platform that can be programmed into exquisite structures through sequence-specific fine tuning of amino acids.
Project description:An amphipathic leucine (L) and lysine (K)-rich ?-helical peptide is multimerized based on helix-loop-helix structures to maximize the penetrating activities. The multimeric LK-based cell penetrating peptides (LK-CPPs) can penetrate cells as protein-fused forms at 100-1000-fold lower concentrations than Tat peptide. The enhanced penetrating activity is increased through multimerization by degrees up to the tetramer level. The multimeric LK-CPPs show rapid cell penetration through macropinocytosis at low nanomolar concentrations, unlike the monomeric LK, which have slower penetrating kinetics at much higher concentrations. The heparan sulfate proteoglycan (HSPG) receptors are highly involved in the rapid internalization of multimeric LK-CPPs. As a proof of concept of biomedical applications, an adipogenic transcription factor, peroxisome proliferator-activated receptor gamma 2 (PPAR-??2), is delivered into preadipocytes, and highly enhanced expression of adipogenic genes at nanomolar concentrations is induced. The multimeric CPPs can be a useful platform for the intracellular delivery of bio-macromolecular reagents that have difficulty with penetration in order to control biological reactions in cells at feasible concentrations for biomedical purposes.
Project description:Protein-protein interactions involve hotspots as small as 4 sequential amino acids. Corresponding tetrapeptides have no structure in water. Here we report linking side chains of amino acids X and Z to form 24 cyclic tetrapeptides, cyclo-[XAAZ]-NH2, and stabilise 14-18 membered rings that mimic different kinds of non-regular secondary structures found in protein hotspots. 2D NMR spectra allowed determination of 3D structures for 14 cyclic tetrapeptides in water. Five formed two (i, i + 3) hydrogen bonds and a beta/gamma (6, 7) or beta (9, 19, 20) turn; eight formed one (i, i + 4) hydrogen bond and twisted into a non-helical (13, 18, 21, 22, 24) or helical (5, 17, 23) alpha turn; one was less structured (15). A beta or gamma turn was favoured for Z = Dab, Orn or Glu due to a ?1 gauche (+) rotamer, while an alpha turn was favoured for Z = Dap (but not X = Dap) due to a gauche (-) rotamer. Surprisingly, an unstructured peptide ARLARLARL could be twisted into a helix when either a helical or non-helical alpha turn (5, 13, 17, 18, 21-24) with Z = Dap was attached to the N-terminus. These structural models provide insights into stability for different turns and twists corresponding to non-regular folds in protein hotspots.
Project description:?3-peptides consisting exclusively of ?3-amino acids adopt a variety of non-natural helical structures and can self-assemble into well-defined hierarchical structures by axial head-to-tail self-assembly resulting in fibrous materials of varying sizes and shapes. To allow control of fiber morphology, a lipid moiety was introduced within a tri-?3-peptide sequence at each of the three amino acid positions and the N-terminus to gain finer control over the lateral assembly of fibers. Depending on the position of the lipid, the self-assembled structures formed either twisted ribbon-like fibers or distinctive multilaminar nanobelts. The nanobelt structures were comprised of multiple layers of peptide fibrils as revealed by puncturing the surface of the nanobelts with an AFM probe. This stacking phenomenon was completely inhibited through changes in pH, indicating that the layer stacking was mediated by electrostatic interactions. Thus, the present study is the first to show controlled self-assembly of these fibrous structures, which is governed by the location of the acyl chain in combination with the 3-point H-bonding motif. Overall, the results demonstrate that the nanostructures formed by the ?3-tripeptide foldamers can be tuned via sequential lipidation of N-acetyl ?3-tripeptides which control the lateral interactions between peptide fibrils and provide defined structures with a greater homogeneous population.
Project description:<h4>Background</h4>Basic cell-penetrating peptides are potential vectors for therapeutic molecules and display antimicrobial activity. The peptide-membrane contact is the first step of the sequential processes leading to peptide internalization and cell activity. However, the molecular mechanisms involved in peptide-membrane interaction are not well understood and are frequently controversial. Herein, we compared the membrane activities of six basic peptides with different size, charge density and amphipaticity: Two cell-penetrating peptides (penetratin and R9), three amphipathic peptides and the neuromodulator substance P.<h4>Methodology/principal findings</h4>Experiments of X ray diffraction, video-microscopy of giant vesicles, fluorescence spectroscopy, turbidimetry and calcein leakage from large vesicles are reported. Permeability and toxicity experiments were performed on cultured cells. The peptides showed differences in bilayer thickness perturbations, vesicles aggregation and local bending properties which form lipidic tubular structures. These structures invade the vesicle lumen in the absence of exogenous energy.<h4>Conclusions/significance</h4>We showed that the degree of membrane permeabilization with amphipathic peptides is dependent on both peptide size and hydrophobic nature of the residues. We propose a model for peptide-induced membrane perturbations that explains the differences in peptide membrane activities and suggests the existence of a facilitated "physical endocytosis," which represents a new pathway for peptide cellular internalization.
Project description:This report focuses on the molecular-level processes and thermodynamics of unfolding of a series of helical peptides using a coarse-grained (CG) molecular model. The CG model was refined to capture thermodynamics and structural changes as a function of temperature for a set of published peptide sequences. Circular dichroism spectroscopy (CD) was used to experimentally monitor the temperature-dependent conformational changes and stability of published peptides and new sequences introduced here. The model predictions were quantitatively or semi-quantitatively accurate in all cases. The simulations and CD results showed that, as expected, in most cases the unfolding of helical peptides is well described by a simply 2-state model, and conformational stability increased with increased length of the helices. A notable exception in a 19-residue helix was when two Ala residues were each replaced with Phe. This stabilized a partly unfolded intermediate state via hydrophobic contacts, and also promoted aggregates at higher peptide concentrations.
Project description:Interfacial systems are at the core of fascinating phenomena in many disciplines, such as biochemistry, soft-matter physics, and food science. However, the parametrization of accurate, reliable, and consistent coarse-grained (CG) models for systems at interfaces remains a challenging endeavor. In the present work, we explore to what extent two independently developed solvent-free CG models of peptides and lipids--of different mapping schemes, parametrization methods, target functions, and validation criteria--can be combined by only tuning the cross-interactions. Our results show that the cross-parametrization can reproduce a number of structural properties of membrane peptides (for example, tilt and hydrophobic mismatch), in agreement with existing peptide-lipid CG force fields. We find encouraging results for two challenging biophysical problems: (i) membrane pore formation mediated by the cooperative action of several antimicrobial peptides, and (ii) the insertion and folding of the helix-forming peptide WALP23 in the membrane.
Project description:Phosphatidylinositol bisphosphate (PIP(2)) is an activator of mammalian inwardly rectifying potassium (Kir) channels. Multiscale simulations, via a sequential combination of coarse-grained and atomistic molecular dynamics, enabled exploration of the interactions of PIP(2) molecules within the inner leaflet of a lipid bilayer membrane with possible binding sites on Kir channels. Three Kir channel structures were investigated: X-ray structures of KirBac1.1 and of a Kir3.1-KirBac1.3 chimera and a homology model of Kir6.2. Coarse-grained simulations of the Kir channels in PIP(2)-containing lipid bilayers identified the PIP(2)-binding site on each channel. These models of the PIP(2)-channel complexes were refined by conversion to an atomistic representation followed by molecular dynamics simulation in a lipid bilayer. All three channels were revealed to contain a conserved binding site at the N-terminal end of the slide (M0) helix, at the interface between adjacent subunits of the channel. This binding site agrees with mutagenesis data and is in the proximity of the site occupied by a detergent molecule in the Kir chimera channel crystal. Polar contacts in the coarse-grained simulations corresponded to long-lived electrostatic and H-bonding interactions between the channel and PIP(2) in the atomistic simulations, enabling identification of key side chains.
Project description:Melanocortin-4 receptor (MC4R) has an important regulatory role in energy homeostasis and food intake. Peptide agonists of the MC4R are characterized by the conserved sequence His(6)-Phe(7)-Arg(8)-Trp(9), which is crucial for their interaction with the receptor. This investigation utilized the covalent attachment approach to identify receptor residues in close proximity to the bound ligand [Nle(4),D-Phe(7)]melanocyte-stimulating hormone (NDP-MSH), thereby differentiating between residues directly involved in ligand binding and those mutations that compromise ligand binding by inducing conformational changes in the receptor. Also, recent X-ray structures of G-protein-coupled receptors were utilized to refine a model of human MC4R in the active state (R(*)), which was used to generate a better understanding of the binding mode of the ligand NDP-MSH at the atomic level. The mutation of residues in the human MC4R--such as Leu106 of extracellular loop 1, and Asp122, Ile125, and Asp126 of transmembrane (TM) helix 3, His264 (TM6), and Met292 (TM7)--to Cys residues produced definitive indications of proximity to the side chains of residues in the core region of the peptide ligand. Of particular interest was the contact between D-Phe(7) on the ligand and Ile125 of TM3 on the MC4R. Additionally, Met292 (TM7) equivalent to Lys(7.45) (Ballesteros numbering scheme) involved in covalently attaching retinal in rhodopsin is shown to be in close proximity to Trp(9). For the first time, the interactions between the terminal regions of NDP-MSH and the receptor are described. The amino-terminus appears to be adjacent to a series of hydrophilic residues with novel interactions at Cys196 (TM5) and Asp189 (extracellular loop 2). These interactions are reminiscent of sequential ligand binding exhibited by the beta(2)-adrenergic receptor, with the former interaction being equivalent to the known interaction involving Ser204 of the beta(2)-adrenergic receptor.
Project description:Amyloid fibrils are ?-sheet-rich protein aggregates that are strongly associated with a variety of neurodegenerative maladies, such as Alzheimer's and Parkinson's diseases. Even if the secondary structure of such fibrils is well characterized, a thorough understanding of their surface organization still remains elusive. Tip-enhanced Raman spectroscopy (TERS) is one of a few techniques that allow the direct characterization of the amino acid composition and the protein secondary structure of the amyloid fibril surface. Herein, we investigated the surfaces of two insulin fibril polymorphs with flat (flat) and left-twisted (twisted) morphology. It was found that the two differ substantially in both amino acid composition and protein secondary structure. For example, the amounts of Tyr, Pro, and His differ, as does the number of carboxyl groups on the respective surfaces, whereas the amounts of Phe and of positively charged amino and imino groups remain similar. In addition, the surface of protofilaments, the precursors of the mature flat and twisted fibrils, was investigated using TERS. The results show substantial differences with respect to the mature fibrils. A correlation of amino acid frequencies and protein secondary structures on the surface of protofilaments and on flat and twisted fibrils allowed us to propose a hypothetical mechanism for the propagation to specific fibril polymorphs. This knowledge can shed a light on the toxicity of amyloids and define the key factors responsible for fibril polymorphism. Finally, this work demonstrates the potential of TERS for the surface characterization of amyloid fibril polymorphs.
Project description:Cell-penetrating peptide (CPP) is a promising cargo for delivering bioactive molecules. In this study, the N terminus of VP1 from chicken anemia virus, designated as CVP1, was found to carry enriched arginine residues with ?-helix. By confocal imaging, flow cytometry and MTT assay, we identified CVP1 as a novel, safe and efficient CPP. CVP1-FITC peptide could entry different types of cells tested with dose dependence, but without cytotoxic effects. Compared with TAT-FITC peptide, the CVP1-FITC peptide showed much higher cell-penetrating activity. Moreover, CVP1 could successfully deliver ?-glycosidase, poly (I:C) and plasmid into HCT116 cells. Inhibitors and temperature sensitivity analysis further indicated that the cell-penetrating activity of CVP1 was based on ATP-dependent and caveolae-mediated endocytosis. All these data demonstrate that CVP1 has efficient cell-penetrating activity and great potential for developing a novel delivery vector.