Richard F. Collins

Self-assembled peptide-based hydrogels as scaffolds for anchorage-dependent cells.

We report here the design of a biomimetic nanofibrous hydrogel as a 3D-scaffold for anchorage-dependent cells. The peptide-based bioactive hydrogel is formed through molecular self-assembly and the building blocks are a mixture of two aromatic short peptide derivatives: Fmoc-FF (Fluorenylmethoxycarbonyl-diphenylalanine) and Fmoc-RGD (arginine-glycine-aspartate) as the simplest self-assembling moieties reported so far for the construction of small-molecule-based bioactive hydrogels. This hydrogel provides a highly hydrated, stiff and nanofibrous hydrogel network that uniquely presents bioactive ligands at the fibre surface; therefore it mimics certain essential features of the extracellular matrix. The RGD sequence as part of the Fmoc-RGD building block plays a dual role of a structural component and a biological ligand. Spectroscopic and imaging analysis using CD, FTIR, fluorescence, TEM and AFM confirmed that FF and RGD peptide sequences self-assemble into beta-sheets interlocked by pi-pi stacking of the Fmoc groups. This generates the cylindrical nanofibres interwoven within the hydrogel with the presence of RGDs in tunable densities on the fibre surfaces. This rapid gelling material was observed to promote adhesion of encapsulated dermal fibroblasts through specific RGD-integrin binding, with subsequent cell spreading and proliferation; therefore it may offer an economical model scaffold to 3D-culture other anchorage-dependent cells for in-vitro tissue regeneration.

Enzyme-assisted self-assembly under thermodynamic control

The production of functional molecular architectures through self-assembly is commonplace in biology, but despite advances, it is still a major challenge to achieve similar complexity in the laboratory. Self-assembled structures that are reproducible and virtually defect free are of interest for applications in three-dimensional cell culture, templating, biosensing and supramolecular electronics. Here, we report the use of reversible enzyme-catalysed reactions to drive self-assembly. In this approach, the self-assembly of aromatic short peptide derivatives provides a driving force that enables a protease enzyme to produce building blocks in a reversible and spatially confined manner. We demonstrate that this system combines three features: (i) self-correction--fully reversible self-assembly under thermodynamic control; (ii) component-selection--the ability to amplify the most stable molecular self-assembly structures in dynamic combinatorial libraries; and (iii) spatiotemporal confinement of nucleation and structure growth. Enzyme-assisted self-assembly therefore provides control in bottom-up fabrication of nanomaterials that could ultimately lead to functional nanostructures with enhanced complexities and fewer defects.

Fmoc-Diphenylalanine Self-Assembly Mechanism Induces Apparent pKa Shifts

We report the effect of pH on the self-assembly process of Fmoc-diphenylalanine (Fmoc-FF) into fibrils consisting of antiparallel beta-sheets, and show that it results in two apparent pKa shifts of approximately 6.4 and approximately 2.2 pH units above the theoretical pKa (3.5). Using Fourier transform infrared (FTIR) spectroscopy, transmission electron microscopy (TEM), wide angle X-ray scattering (WAXS), and oscillatory rheology, these two transitions were shown to coincide with significant structural changes. An entangled network of flexible fibrils forming a weak hydrogel dominates at high pH, while nongelling flat rigid ribbons form at intermediate pH values. Overall, this study provides further understanding of the self-assembly mechanism of aromatic short peptide derivatives.

3-D structural and functional characterization of the purified KATP channel complex Kir6.2-SUR1.

ATP‐sensitive potassium (KATP) channels conduct potassium ions across cell membranes and thereby couple cellular energy metabolism to membrane electrical activity. Here, we report the heterologous expression and purification of a functionally active KATP channel complex composed of pore‐forming Kir6.2 and regulatory SUR1 subunits, and determination of its structure at 18 Å resolution by single‐particle electron microscopy. The purified channel shows ATP‐ase activity similar to that of ATP‐binding cassette proteins related to SUR1, and supports Rb+ fluxes when reconstituted into liposomes. It has a compact structure, with four SUR1 subunits embracing a central Kir6.2 tetramer in both transmembrane and cytosolic domains. A cleft between adjacent SUR1s provides a route by which ATP may access its binding site on Kir6.2. The nucleotide‐binding domains of adjacent SUR1 appear to interact, and form a large docking platform for cytosolic proteins. The structure, in combination with molecular modelling, suggests how SUR1 interacts with Kir6.2.

The 3D structure of a periplasm-spanning platform required for assembly of group 1 capsular polysaccharides in Escherichia coli

Capsular polysaccharides (CPSs) are essential virulence determinants of many pathogenic bacteria. Escherichia coli group 1 CPSs provide paradigms for widespread surface polysaccharide assembly systems in Gram-negative bacteria. In these systems, complex carbohydrate polymers must be exported across the periplasm and outer membrane to the cell surface. Group 1 CPS export requires oligomers of the outer membrane protein, Wza, for translocation across the outer membrane. Assembly also depends on Wzc, an inner membrane tyrosine autokinase known to regulate export and synthesis of group 1 CPS. Here, we provide a structural view of a complex comprising Wzc and Wza that spans the periplasm, connecting the inner and outer membranes. Examination of transmembrane sections of the complex suggests that the periplasm is compressed at the site of complex formation. An important feature of CPS production is the coupling of steps involved in biosynthesis and export. We propose that the Wza–Wzc complex provides the structural and regulatory core of a larger macromolecular machine. We suggest a mechanism by which CPS may move from the periplasm through the outer membrane.

An investigation of the conductivity of peptide nanotube networks prepared by enzyme-triggered self-assembly

We demonstrate that nanotubular networks formed by enzyme-triggered self-assembly of Fmoc-L3 (9-fluorenylmethoxycarbonyl-tri-leucine) show significant charge transport. FT-IR, fluorescence spectroscopy and wide angle X-ray scattering (WAXS) data confirm formation of beta-sheets that are locked together viapi-stacking interactions. Molecular dynamics simulations confirmed the pi-pi stacking distance between fluorenyl groups to be 3.6-3.8 A. Impedance spectroscopy demonstrated that the nanotubular xerogel networks possess minimum sheet resistances of 0.1 MOmega/sq in air and 500 MOmega/sq in vacuum (pressure: 1.03 mbar) at room temperature, with the conductivity scaling linearly with the mass of peptide in the network. These materials may provide a platform to interface biological components with electronics.

Structure of LIMP-2 provides functional insights with implications for SR-BI and CD36.

Members of the CD36 superfamily of scavenger receptor proteins are important regulators of lipid metabolism and innate immunity. They recognize normal and modified lipoproteins, as well as pathogen-associated molecular patterns. The family consists of three members: SR-BI (which delivers cholesterol to the liver and steroidogenic organs and is a co-receptor for hepatitis C virus), LIMP-2/LGP85 (which mediates lysosomal delivery of β-glucocerebrosidase and serves as a receptor for enterovirus 71 and coxsackieviruses) and CD36 (a fatty-acid transporter and receptor for phagocytosis of effete cells and Plasmodium-infected erythrocytes). Notably, CD36 is also a receptor for modified lipoproteins and β-amyloid, and has been implicated in the pathogenesis of atherosclerosis and of Alzheimer’s disease. Despite their prominent roles in health and disease, understanding the function and abnormalities of the CD36 family members has been hampered by the paucity of information about their structure. Here we determine the crystal structure of LIMP-2 and infer, by homology modelling, the structure of SR-BI and CD36. LIMP-2 shows a helical bundle where β-glucocerebrosidase binds, and where ligands are most likely to bind to SR-BI and CD36. Remarkably, the crystal structure also shows the existence of a large cavity that traverses the entire length of the molecule. Mutagenesis of SR-BI indicates that the cavity serves as a tunnel through which cholesterol(esters) are delivered from the bound lipoprotein to the outer leaflet of the plasma membrane. We provide evidence supporting a model whereby lipidic constituents of the ligands attached to the receptor surface are handed off to the membrane through the tunnel, accounting for the selective lipid transfer characteristic of SR-BI and CD36.

Analysis of the PilQ Secretin from Neisseria meningitidis by Transmission Electron Microscopy Reveals a Dodecameric Quaternary Structure

PilQ is a member of the secretin family of outer membrane proteins and is specifically involved in secretion of type IV pili in Neisseria meningitidis, Neisseria gonorrhoeae, and Pseudomonas aeruginosa. The quaternary structure of PilQ from N. meningitidis was analyzed by transmission electron microscopy by using a negative stain. Single particle averaging was carried out with a total data set of 650 individual particles, which produced a projection map generated from 296 particles at an estimated resolution of 2.6 nm. Oligomeric PilQ adopts a donut-like structure with an external ring that is 16.5 nm in diameter surrounding a central cavity that is 6.5 nm in diameter. Self-rotation and power spectrum analysis demonstrated the presence of 12-fold rotational symmetry, showing that PilQ is organized as a ring of 12 identical subunits. A model of the type IV meningococcal pilus fiber, based on the X-ray crystal structure of the N. gonorrhoeae pilin subunit, fitted neatly into the cavity, demonstrating how PilQ could serve as a channel for the growing pilus fiber.

Exploiting enzymatic (reversed) hydrolysis in directed self-assembly of peptide nanostructures

Enzyme-catalyzed reactions can be exploited to control molecular self-assembly under physiological conditions by converting nonassembling precursors into self-assembly building blocks. Two complementary approaches based on aromatic short-peptide derivatives that form molecular hydrogels are demonstrated. Firstly, it is shown that esterase-directed self assembly via hydrolysis of hydrophobic N-(fluorenyl-9-methoxycarbonyl) (Fmoc)-peptide methyl esters give rise to formation of transparent hydrogels composed of defined peptide nanotubes. The internal and external diameters of these tubes are highly tunable, depending on the amino acid composition and chain length of the building blocks. Secondly, protease-directed self-assembly of Fmoc-peptide esters is achieved via amide-bond formation (reversed hydrolysis) for combinations of Fmoc-threonine and leucine/phenylalanine methyl esters, producing fibrous hydrogels. Upon treatment with an esterase, these systems revert back to solution, thus providing a two-stage solution-gel-solution transition.

Self-assembly and gelation properties of α-helix versus β-sheet forming peptides

We have investigated the self-assembly and gelation properties of a set of four octa-peptides: AEAEAKAK, AEAKAEAK, FEFEFKFK and FEFKFEFK. The phenylalanine based peptides adopt β-sheet conformations in solution and the alanine based peptides form α-helices. No self-assembly in solution was observed for AEAKAEAK but AEAEAKAK was found to self-assemble forming thick, rigid fibres with a diameter of ∼6 nm. These fibres were composed of two fibrils aggregating side by side to form “pearl-necklace” morphologies. No gelation was observed for AEAEAKAK in the concentration range investigated (0 to 100 mg ml−1). In contrast, both phenylalanine based peptides were found to self-assemble in solution and to form hydrogels at an initial concentration of ∼8 mg ml−1. Similar morphologies were observed for both peptides corresponding to a relatively homogeneous dense network of semi-flexible fibres with a mesh size of ∼15 to 30 nm depending on the concentration. The fibre diameter was found to be ∼4 nm in good agreement with models found in the literature. TEM micrographs clearly showed that these fibres have a helicoidal or twisted structure. Comparison of TEM with AFM data highlighted the influence of substrate chemistry on the macromolecular assembly of small peptides. In contrast small angle neutron scattering (SANS) approaches, which allow for the probing of hydrogel morphology and structure without the need for sample preparation on solid substrates, provide vital data on hydrogel morphology in solution.