No abstract

Specific protein-protein interactions are crucial for virtually all biological processes. 1 Naturally occurring surfaces that mediate protein-protein interactions usually contain either elements of polar specificity, for example hydrogen bonding or salt bridges, or complementary hydrophobic patches that contain side chains that maximize van der Waals interactions. 2 We, 3 and others 4 have recently described a new type of protein-protein interaction interface that could potentially be both hydrophobic and lipophobic. This is accomplished by the introduction of nonproteinogenic, fluorine-containing amino acids. 5,6 Reported here is the design, synthesis, and programmed self-sorting of peptide systems with orthogonally miscible hydrocarbon and fluorous (highly fluorinated) cores.Peptides H and F are equipped with N-terminal cysteine residues and were designed to form parallel homodimeric coiled coil assemblies. 7 These peptides have an identical sequence except that all seven of the core leucine residues in H have been replaced by 5,5,5,5′,5′,5′-R-S-hexafluoroleucine in F (Figure 1), shielding 28 trifluoromethyl groups from aqueous solvent in the canonical fluorinated dimer. Hexafluoroleucine was synthesized according to a recently documented procedure. 8 The peptides were assembled on 4-methylbenzhydrylamine (MBHA) resin according to the in situ neutralization protocol for t-Boc peptide synthesis as described previously 9 and purified by reverse-phase HPLC. Purity of the peptides was confirmed by analytical HPLC and MALDI mass spectrometry. H and F are designed to form parallel coiled coil structures due to unfavorable interhelical electrostatic interactions in the antiparallel arrangements. 10 Furthermore, a single polar residue Asn14 was incorporated in the hydrophobic

PEGylated liposomes are attractive pharmaceutical nanocarriers; however, literature reports of ligand-targeted nanoparticles have not consistently shown successful results. Here, we employed a multifaceted synthetic strategy to prepare peptide-targeted liposomal nanoparticles with high purity, reproducibility, and precisely controlled stoichiometry of functionalities to evaluate the role of liposomal PEG coating, peptide EG-linker length, and peptide valency on cellular uptake in a systematic manner. We analyzed these parameters in two distinct disease models where the liposomes were functionalized with either HER2- or VLA-4-antagonistic peptides to target HER2-overexpressing breast cancer cells or VLA-4-overexpressing myeloma cells, respectively. When targeting peptides were tethered to nanoparticles with an EG45 (∼PEG2000) linker in a manner similar to a more traditional formulation, their cellular uptake was not enhanced compared to non-targeted versions regardless of the liposomal PEG coating used. Conversely, reduction of the liposomal PEG to PEG350 and the peptide linker to EG12 dramatically enhanced cellular uptake by ∼9 fold and ∼100 fold in the breast cancer and multiple myeloma cells, respectively. Uptake efficiency reached a maximum and a plateau with ∼2% peptide density in both disease models. Taken together, these results demonstrate the significance of using the right design elements such as the appropriate peptide EG-linker length in coordination with the appropriate liposomal PEG coating and optimal ligand density in efficient cellular uptake of liposomal nanoparticles.

The design, synthesis, and structural characterization of a highly fluorinated peptide system based on the coiled coil region of the yeast transcription factor GCN4 is described. All four leucine residues (a position) and three valine residues (d position) were replaced by the unnatural amino acids 5,5,5-trifluoroleucine and 4,4,4-trifluorovaline, respectively. The peptide is highly alpha-helical at low micromolar concentrations as judged by circular dichroism spectra, sediments as a dimeric species in the 5-30 microM concentration range, and exhibits a dimer melting temperature that is 15 degrees C higher than a control peptide with a hydrocarbon core. Furthermore, the apparent free energy of unfolding as calculated from guanidinium hydrochloride denaturation experiments is larger by approximately 1.0 kcal/mol for the fluorinated peptide than its hydrocarbon counterpart. We conclude that additional stability is derived from sequestering the more hydrophobic trifluoromethyl groups from aqueous solvent. These studies introduce a new paradigm in the design of molecular self-assembling systems, one based on orthogonal solubility properties of liquid phases.

The amino acid substitution or post-translational modification of a cytosolic protein can cause unpredictable changes to its electrophoretic mobility during SDS-PAGE. This type of ''gel shifting'' has perplexed biochemists and biologists for decades. We identify a mechanism for ''gel shifting'' that predominates among a set of ALS (amyotrophic lateral sclerosis) mutant hSOD1 (superoxide dismutase) proteins, post-translationally modified hSOD1 proteins, and homologous SOD1 proteins from different organisms. By first comparing how 39 amino acid substitutions throughout hSOD1 affected SDS-PAGE migration, we found that substitutions that caused gel shifting occurred within a single polyacidic domain (residues~80-101), and were nonisoelectric. Substitutions that decreased the net negative charge of domain 80-101 increased migration; only one substitution increased net negative charge and slowed migration. Capillary electrophoresis, circular dichroism, and size exclusion chromatography demonstrated that amino acid substitutions increase migration during SDS-PAGE by promoting the binding of three to four additional SDS molecules, without significantly altering the secondary structure or Stokes radius of hSOD1-SDS complexes. The high negative charge of domain 80-101 is required for SOD1 gel shifting: neutralizing the polyacidic domain (via chimeric mouse-human SOD1 fusion proteins) inhibited amino acid substitutions from causing gel shifting. These results demonstrate that the pattern of gel shifting for mutant cytosolic proteins can be used to: (i) identify domains in the primary structure that control interactions between denatured cytosolic proteins and SDS and (ii) identify a predominant chemical mechanism for the interaction (e.g., hydrophobic vs. electrostatic).

This paper describes a synthetic dimer of carbonic anhydrase, and a series of bivalent sulfonamide ligands with different lengths (25 to 69 Å between the ends of the fully extended ligands), as a model system to use in examining the binding of bivalent antibodies to antigens. Assays based on analytical ultracentrifugation and fluorescence binding indicate that this system forms cyclic, noncovalent complexes with a stoichiometry of one bivalent ligand to one dimer. This dimer binds the series of bivalent ligands with low picomolar avidities (Kdavidity = 3 – 40 pM). A structurally analogous monovalent ligand binds to one active site of the dimer with Kdmono = 16 nM. The bivalent association is thus significantly stronger (Kdmono/Kdavidity ranging from ~500 to 5000 unitless) than the monovalent association. We infer from these results, and by comparison of these results to previous studies, that bivalency in antibodies can lead to associations much tighter than monovalent associations (although the observed bivalent association is much weaker than predicted from the simplest level of theory—predicted Kdavidity of ~ 0.002 pM and Kdmono/Kdavidity ~ 8 × 106 unitless).

Cisplatin is a first line chemotherapy for most types of cancer. However, its use is dose-limited due to severe nephrotoxicity. Here we report the rational engineering of a novel nanoplatinate inspired by the mechanisms underlying cisplatin bioactivation. We engineered a novel polymer, glucosamine-functionalized polyisobutylene-maleic acid, where platinum (Pt) can be complexed to the monomeric units using a monocarboxylato and an O → Pt coordinate bond. We show that at a unique platinum to polymer ratio, this complex self-assembles into a nanoparticle, which releases cisplatin in a pH-dependent manner. The nanoparticles are rapidly internalized into the endolysosomal compartment of cancer cells, and exhibit an IC50 (4.25 AE 0.16 μM) comparable to that of free cisplatin (3.87 AE 0.37 μM), and superior to carboplatin (14.75 AE 0.38 μM). The nanoparticles exhibited significantly improved antitumor efficacy in terms of tumor growth delay in breast and lung cancers and tumor regression in a K-ras LSL∕þ ∕Pten fl∕fl ovarian cancer model. Furthermore, the nanoparticle treatment resulted in reduced systemic and nephrotoxicity, validated by decreased biodistribution of platinum to the kidney as quantified using inductively coupled plasma spectroscopy. Given the universal need for a better platinate, we anticipate this coupling of nanotechnology and structureactivity relationship to rationally reengineer cisplatin could have a major impact globally in the clinical treatment of cancer.chemotherapy | nanomedicine | cancer

Control of structure and function in membrane proteins remains a formidable challenge. We report here a new design paradigm for the self-assembly of protein components in the context of nonpolar environments of biological membranes. An incrementally staged assembly process relying on the unique properties of fluorinated amino acids was used to drive transmembrane helixhelix interactions. In the first step, hydrophobic peptides partitioned into micellar lipids. Subsequent phase separation of simultaneously hydrophobic and lipophobic fluorinated helical surfaces fueled spontaneous self-assembly of higher order oligomers. The creation of these ordered transmembrane protein ensembles is supported by gel electrophoresis, circular dichroism spectroscopy, equilibrium analytical ultracentrifugation, and fluorescence resonance energy transfer.