0000000000021495
AUTHOR
Frank Depoix
Pentafluorophenyl Ester-based Polymersomes as Nanosized Drug-Delivery Vehicles
In this work, activated ester chemistry is employed to synthesize biocompatible and readily functionalizable polymersomes. Via aminolysis of pentafluorophenyl methacrylate-based precursor polymers, an N-(2-hydroxypropyl) methacrylamide (HPMA)-analog hydrophilic block is obtained. The precursor polymers can be versatile functionalized by simple addition of suitable primary amines during aminolysis as demonstrated using a fluorescent dye. Vesicle formation is proven by cryoTEM and light scattering. High encapsulation efficiencies for hydrophilic cargo like siRNA are achieved using dual centrifugation and safe encapsulation is demonstrated by gel electrophoresis. In vitro studies reveal low cy…
Nautilus pompilius Hemocyanin: 9 Å Cryo-EM Structure and Molecular Model Reveal the Subunit Pathway and the Interfaces between the 70 Functional Units
Hemocyanins are giant extracellular oxygen carriers in the hemolymph of many molluscs. Nautilus pompilius (Cephalopoda) hemocyanin is a cylindrical decamer of a 350 kDa polypeptide subunit that in turn is a "pearl-chain" of seven different functional units (FU-a to FU-g). Each globular FU has a binuclear copper centre that reversibly binds one O(2) molecule, and the 70-FU decamer is a highly allosteric protein. Its primary structure and an 11 A cryo-electron microscopy (cryo-EM) structure have recently been determined, and the crystal structures of two related FU types are available in the databanks. However, in molluscan hemocyanin, the precise subunit pathway within the decamer, the inter…
Limulus polyphemus Hemocyanin: 10 Å Cryo-EM Structure, Sequence Analysis, Molecular Modelling and Rigid-body Fitting Reveal the Interfaces Between the Eight Hexamers
Abstract The blue copper protein hemocyanin from the horseshoe crab Limulus polyphemus is among the largest respiratory proteins found in nature (3.5 MDa) and exhibits a highly cooperative oxygen binding. Its 48 subunits are arranged as eight hexamers (1×6mers) that form the native 8×6mer in a nested hierarchy of 2×6mers and 4×6mers. This quaternary structure is established by eight subunit types (termed I, IIA, II, IIIA, IIIB, IV, V, and VI), of which only type II has been sequenced. Crystal structures of the 1×6mer are available, but for the 8×6mer only a 40 A 3D reconstruction exists. Consequently, the structural parameters of the 8×6mer are not firmly established, and the molecular inte…
Alhydrogel® adjuvant, ultrasonic dispersion and protein binding: A TEM and analytical study
Aluminium-based vaccine adjuvants have been in use since the 1920s. Aluminium hydroxide (alum) that is the chemical basis of Alhydrogel, a widely used adjuvant, is a colloid that binds proteins to the particular surface for efficient presentation to the immune system during the vaccination process. Using conventional TEM and cryo-TEM we have shown that Alhydrogel can be finely dispersed by ultrasonication of the aqueous suspension. Clusters of ultrasonicated aluminium hydroxide micro-fibre crystals have been produced (∼ 10-100 nm), that are significantly smaller than those present the untreated Alhydrogel (∼ 2-12 μm). However, even prolonged ultrasonication did not produce a homogenous susp…
Allosterism of Nautilus pompilius hemocyanin as deduced from 8 Å cryo-EM structures obtained under oxy and deoxy conditions
Hemocyanins are the blue copper-containing respiratory proteins of many molluscs. Nautilus pompilius hemocyanin (NpH) is a cylindrical decamer composed of ten copies of a 350 kDa polypeptide subunit, in turn consisting of seven O2-binding functional units (FUs, termed NpH-a to NpH-g). Ten copies of the subunit segment NpH-a to NpH-f form the cylinder wall (ca. 35 nm in diameter), whereas the ten copies of NpH-g build the internal collar. Recently we published a 9A cryo-EM structure and molecular model of NpH that solved the principal architecture of this protein [1]. Hemocyanins are highly allosteric, and the cooperativity of oxygen binding should be transferred between functional units by …
Lipid Bilayer Interactions of Peptidic Supramolecular Polymers and Their Impact on Membrane Permeability and Stability.
The synthesis and physicochemical characterization of supramolecular polymers with tunable assembly profiles offer exciting opportunities, involving the development of new biomedical carriers. Because synthetic nanocarriers aim to transport substances across or toward cellular membranes, we evaluated the interactions of amphiphilic peptide-based supramolecular polymers with lipid bilayers. Here, we focused on nanorod-like supramolecular polymers, obtained from two C3-symmetric dendritic peptide amphiphiles with alternating Phe/His sequences, equipped with a peripheral tetraethylene glycol dendron (C3-PH) or charged ethylenediamine end groups (C3-PH+). Triggered by pH changes, these amphiphi…
10-A cryoEM structure and molecular model of the Myriapod (Scutigera) 6x6mer hemocyanin:understanding a giant oxygen transport protein
Oxygen transport in Myriapoda is maintained by a unique 6x6mer hemocyanin, that is, 36 subunits arranged as six hexamers (1x6mers). In the sluggish diplopod Spirostreptus, the 1x6mers seem to operate as almost or fully independent allosteric units (h approximately 1.3; P(50) approximately 5 torr), whereas in the swift centipede Scutigera, they intensively cooperate allosterically (h approximately 10; P(50) approximately 50 torr). Here, we show the chemomechanical basis of this differential behavior as deduced from hybrid 6x6mer structures, obtained by single-particle cryo-electron microscopy of the Scutigera 6x6mer (10.0 A resolution according to the 0.5 criterion) and docking of homology-m…
Functionalization of Active Ester-Based Polymersomes for Enhanced Cell Uptake and Stimuli-Responsive Cargo Release
Poly(2,3-dihydroxypropyl methacrylamide) (P(DHPMA))-based amphiphilic block copolymers have recently proven to form polymer vesicles (polymersomes). In this work, we further expand their potential by incorporating (i) units for pH-dependent disintegration into the hydrophobic membrane and (ii) mannose as targeting unit into the hydrophilic block. This last step relies on the use of an active ester prepolymer. We confirm the stability of the polymersomes against detergents like Triton X-100 and their low cytotoxicity. The incorporation of 2-(2,2-dimethyl-1,3-dioxolane-4-yl)ethyl methacrylate into the hydrophobic block (lauryl methacrylate) allows a pH-responsive disintegration for cargo rele…
8 Å cryo-TEM and single particle analysis of Nautilus pompilius hemocyanin under two states of oxygenation
Extended abstract of a paper presented at MC 2007, 33rd DGE Conference in Saarbrücken, Germany, September 2 – September 7, 2007
9 Å cryo-EM structure and molecular model of a gastropod hemocyanin didecamer (KLH1) reveals the architecture of the asymmetric collar
Hemocyanins are blue copper proteins that transport oxygen in the hemolymph of many arthropods and molluscs. Molluscan hemocyanins are decamers, didecamers or multidecamers of a 350–400 kDa polypeptide subunit that is subdivided into seven or eight different functional units (FUs, each with a single copper active site). The quaternary structure is a semi-hollow cylinder consisting of a wall and a collar. Recently, we published a 9 A cryo-EM structure and molecular model of a cephalopod hemocyanin decamer (NpH, from Nautilus pompilius) that answered many hitherto unsolved questions concerning the quaternary structure of molluscan hemocyanin. Notably, it revealed the twisted pathway of the 10…
8 Å cryo-EM structure of the giant hemoglobin from the planorbid snail Biomphalaria glabrata
Until 2006, snail red hemoglobin remained a phylogenetic enigma because it occurs quite isolated in a single gastropod family, the Planorbidae, whereas all other gastropods use blue hemocyanin as a respiratory protein (for recent cryo-EM of hemocyanin, see [1,2]). Moreover, sequence data on this snail hemoglobin were completely lacking. In 2006, our group published the complete cDNA and predicted amino acid sequence of two Biomphalaria glabrata hemoglobin polypeptides, termed BgHb1 and BgHb2 [3]. (Biomphalaria is intermediate host of the human parasite Schistosoma mansoni that causes Bilharziosis.) Resembling pearl-chains, both polypeptide subunits encompass 13 different, cysteine-free glob…
A new automated plunger for cryopreparation of proteins in defined - even oxygen free - atmospheres
We study the structure and function of hemocyanins. They are giant extracellular oxygen carriers in the hemolymph of many molluscs and arthropods. Since some of these blue, copper-containing proteins show the highest cooperativity in nature (h = 10), one of our goals is to understand the chemomechanical interaction between the different substructures during allosteric oxygen binding.
Against Expectations: Unassisted RNA Adsorption onto Negatively Charged Lipid Bilayers
The composition and physicochemical properties of biological membranes can be altered by diverse membrane integral and peripheral proteins as well as by small molecules, natural and synthetic. Diverse oligonucleotides have been shown to electrostatically interact with cationic and bivalent ion loaded zwitterionic liposomes, leading to the formation of oligonucleotide-liposome aggregates. However, interaction of RNAs with other membrane surfaces remains ill understood. We used the nonnatural RNA10 to investigate RNA binding to anionic and net-uncharged membrane surfaces. RNA10 had initially been selected in a screen for nonnatural RNA motives that bind to phosphatidylcholine liposomes in the…
The structure of gas-filled n-butyl-2-cyanoacrylate (BCA) polymer particles
Abstract The structure of gas-filled poly-[n-butyl-2-cyanoacrylate] (BCA) particles has been demonstrated by negative staining with uranyl acetate, platinum-carbon shadowing of air-dried material and thin sectioning of the aqueous suspension of BCA particles, embedded in water-soluble melamine resin. The polymer shell of the hollow particles possesses a globular outer surface and a smoother inner surface.
A synthetic biology approach for the fabrication of functional (fluorescent magnetic) bioorganic–inorganic hybrid materials in sponge primmorphs
During evolution, sponges (Porifera) have honed the genetic toolbox and biosynthetic mechanisms for the fabrication of siliceous skeletal components (spicules). Spicules carry a protein scaffold embedded within biogenic silica (biosilica) and feature an amazing range of optical, structural, and mechanical properties. Thus, it is tempting to explore the low-energy synthetic pathways of spiculogenesis for the fabrication of innovative hybrid materials. In this synthetic biology approach, the uptake of multifunctional nonbiogenic nanoparticles (fluorescent, superparamagnetic) by spicule-forming cells of bioreactor-cultivated sponge primmorphs provides access to spiculogenesis. The ingested nan…
Comparative 11A structure of two molluscan hemocyanins from 3D cryo-electron microscopy
Abstract Hemocyanins are giant extracellular proteins that transport oxygen in the hemolymph of many molluscs. Molluscan hemocyanins are cylindrical decamers or didecamers of a 350–400 kDa subunit that contains seven or eight different covalently linked globular functional units (FUs), arranged in a linear manner. Each FU carries a single copper active site and reversibly binds one dioxygen molecule. As a consequence, the decamer can carry up to 70 or 80 O 2 molecules. Although complete sequence information is now available from several molluscan hemocyanins, many details of the quaternary structure are still unclear, including the topology of the 10 subunits within the decamer. Here we sho…