Cell-penetrating peptides (CPPs), such as the HIV TAT peptide, are able to translocate across cellular membranes efficiently. A number of mechanisms, from direct entry to various endocytotic mechanisms (both receptor independent and receptor dependent), have been observed but how these specific amino acid sequences accomplish these effects is unknown. We show how CPP sequences can multiplex interactions with the membrane, the actin cytoskeleton, and cell-surface receptors to facilitate different translocation pathways under different conditions. Using "nunchuck" CPPs, we demonstrate that CPPs permeabilize membranes by generating topologically active saddle-splay ("negative Gaussian") membrane curvature through multidentate hydrogen bonding of lipid head groups. This requirement for negative Gaussian curvature constrains but underdetermines the amino acid content of CPPs. We observe that in most CPP sequences decreasing arginine content is offset by a simultaneous increase in lysine and hydrophobic content. Moreover, by densely organizing cationic residues while satisfying the above constraint, TAT peptide is able to combine cytoskeletal remodeling activity with membrane translocation activity. We show that the TAT peptide can induce structural changes reminiscent of macropinocytosis in actin-encapsulated giant vesicles without receptors.protein transduction domain | polyarginine | peptide-lipid interactions | pore-forming peptide | antimicrobial peptide C ell-penetrating peptides (CPPs) are effective intracellular delivery systems (1-5). These peptides are usually short (<20 amino acids) and cationic. Examples include the TAT peptide from HIV, antennapedia (ANTP) from Drosophila, and even simple polyarginines. Although unique molecular architectures incorporating CPPs have been designed for drug delivery (3, 6-8), the molecular mechanisms of cellular entry, and the relations between them, are not well understood. Different uptake mechanisms have been proposed for CPPs (9). Cell-based assays have indicated that multiple endocytotic pathways are involved (10-15). In addition to these, CPPs are also capable of direct entry mechanisms* (17-20). In general, cell-penetrating activity of CPPs has proven to be difficult to eliminate completely using a specific set of conditions (3,12,21), suggesting the existence of multiple mechanisms. A unified understanding of CPPs, which is currently lacking, must engage why the same sequence can readily activate the qualitatively distinct outcomes.How do relatively simple molecules like HIV TAT peptide facilitate mechanisms as different as direct translocation, and multiple endocytotic processes? Rather than debate priority between mechanisms, we focus on the physical chemistry of what these different mechanisms and CPPs have in common. Here, we show how the TAT peptide can multiplex different interactions with the same sequence, thus interacting with the membrane, the actin cytoskeleton, and specific receptors to produce multiple pathways of translocation under different condition...

We have developed a facile, scalable method for preparation of enzyme responsive copolypeptide vesicles that requires no protecting groups or expensive components. We designed

We describe the preparation and assembly of glycosylated amphiphilic diblock copolypeptides, where the hydrophilic glycosylated segments adopt either a-helical or disordered conformations. In this study, glycosylated amphiphilic diblock copolypeptides were prepared using poly(L-leucine), poly(L), as the hydrophobic segment, and poly(a-D-galactopyranosyl-L-lysine), poly(a-gal-K), or poly(a-D-galactopyranosyl-L-cysteine sulfone), poly(a-gal-C O2), as the hydrophilic segment. The poly(a-gal-K) and poly(a-gal-C O2) segments are known to be fully a-helical (>90% at 20 C) and fully disordered in water, respectively. We found that block copolypeptides containing galactosylated hydrophilic segments of either a-helical or disordered conformation give different assembly morphologies, where the disordered glycopolypeptide segments favor vesicle formation and also present sugar residues that can bind to biological targets.

The evolution of hydrogen bonding with temperature in three copoly(ether-urea)s with no phase separation has been studied by Fourier transform infrared spectroscopy (FT-IR) in the region of the carbonyl absorption of the urea groups. Assignment of the bands was made by supposing the existence of chains of urea groups linked by bifurcated hydrogen bonds. Quantum mechanical calculations of these chains fitted qualitatively with the experimental results. Dynamic mechanical and calorimetric experiments showed that the apparition of the band related to the "internal" urea groups of the chains produces a decrease in the glass transition of the polyether moiety of the copolymer and that prior to it a transition that we related to the glass transition of the bis(ureaphenyl)methane units takes place.X

The design, synthesis, and self-assembly of the first dual hydrophilic triblock copolypeptide vesicles, R(H)(m)E(n)L(o) and K(P)(m)R(H)(n)L(o), is reported. Variation of the two distinct hydrophilic domains is used to tune cellular interactions without disrupting the self-assembled structure. The aqueous self-assemblies of these triblock copolypeptides in water are characterized using microscopy and DLS. Cell culture studies are used to evaluate cytotoxicity as well as intracellular uptake of the vesicles. The ability of polypeptides to incorporate ordered chain conformations that direct self-assembly, combined with the facile preparation of functional, multiblock copolypeptide sequences of defined lengths, allow the design of vesicles attractive for development as drug carriers.