Self-assembling layers created by membrane proteins on gold

Orla Protein Technologies Ltd, Nanotechnology Centre, Newcastle upon Tyne NE1 7RU, UK.
Biochemical Society Transactions (Impact Factor: 3.19). 07/2007; 35(Pt 3):522-6. DOI: 10.1042/BST0350522
Source: PubMed


Membrane systems are based on several types of organization. First, amphiphilic lipids are able to create monolayer and bilayer structures which may be flat, vesicular or micellar. Into these structures membrane proteins can be inserted which use the membrane to provide signals for lateral and orientational organization. Furthermore, the proteins are the product of highly specific self-assembly otherwise known as folding, which mostly places individual atoms at precise places in three dimensions. These structures all have dimensions in the nanoscale, except for the size of membrane planes which may extend for millimetres in large liposomes or centimetres on planar surfaces such as monolayers at the air/water interface. Membrane systems can be assembled on to surfaces to create supported bilayers and these have uses in biosensors and in electrical measurements using modified ion channels. The supported systems also allow for measurements using spectroscopy, surface plasmon resonance and atomic force microscopy. By combining the roles of lipids and proteins, highly ordered and specific structures can be self-assembled in aqueous solution at the nanoscale.

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Available from: Stephen Andrew Holt, Oct 02, 2015
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    • "Peptides and proteins with thiol-containing cysteine residues in their amino acid sequence can be included into mixed SAM on gold creating functional protein arrays [3,4] and membrane proteins are especially suitable since they naturally assemble into layers [5]. Membrane proteins with a β-barrel structure are particularly well suited to immobilisation as they can be specifically immobilised to a gold surface with a controlled orientation, retain their structure and function once immobilised and can be engineered to perform different functions [6]. "
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    ABSTRACT: Bacterial outer membrane proteins, along with a filling lipid molecule can be modified to form stable self-assembled monolayers on gold. The transmembrane domain of Escherichia coli outer membrane protein A has been engineered to create a scaffold protein to which functional motifs can be fused. In earlier work we described the assembly and structure of an antibody-binding array where the Z domain of Staphylococcus aureus protein A was fused to the scaffold protein. Whilst the binding of rabbit polyclonal immunoglobulin G (IgG) to the array is very strong, mouse monoclonal IgG dissociates from the array easily. This is a problem since many immunodiagnostic tests rely upon the use of mouse monoclonal antibodies. Here we describe a strategy to develop an antibody-binding array that will bind mouse monoclonal IgG with lowered dissociation from the array. A novel protein consisting of the scaffold protein fused to two pairs of Z domains separated by a long flexible linker was manufactured. Using surface plasmon resonance the self-assembly of the new protein on gold and the improved binding of mouse monoclonal IgG were demonstrated.
    International Journal of Molecular Sciences 12/2011; 12(8):5157-67. DOI:10.3390/ijms12085157 · 2.86 Impact Factor
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    • "Our approach has been to design and fabricate protein scaffolds that can display protein ligands on self-assembled monolayers (SAM) in a controlled and reproducible way [8,9]. This work uses a TolAIII-fusion expression system to provide the engineered protein at high yields and solubility [10]. "
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    ABSTRACT: Background The interfacial molecular mechanisms that regulate mammalian cell growth and differentiation have important implications for biotechnology (production of cells and cell products) and medicine (tissue engineering, prosthetic implants, cancer and developmental biology). We demonstrate here that engineered protein motifs can be robustly displayed to mammalian cells in vitro in a highly controlled manner using a soluble protein scaffold designed to self assemble on a gold surface. Results A protein was engineered to contain a C-terminal cysteine that would allow chemisorption to gold, followed by 12 amino acids that form a water soluble coil that could switch to a hydrophobic helix in the presence of alkane thiols. Bioactive motifs from either bone morphogenetic protein-2 or osteopontin were added to this scaffold protein and when assembled on a gold surface assessed for their ability to influence cell function. Data demonstrate that osteoblast adhesion and short-term responsiveness to bone morphogenetic protein-2 is dependent on the surface density of a cell adhesive motif derived from osteopontin. Furthermore an immobilised cell interaction motif from bone morphogenetic protein supported bone formation in vitro over 28 days (in the complete absence of other osteogenic supplements). In addition, two-dimensional patterning of this ligand using a soft lithography approach resulted in the spatial control of osteogenesis. Conclusion These data describe an approach that allows the influence of immobilised protein ligands on cell behaviour to be dissected at the molecular level. This approach presents a durable surface that allows both short (hours or days) and long term (weeks) effects on cell activity to be assessed. This widely applicable approach can provide mechanistic insight into the contribution of immobilised ligands in the control of cell activity.
    BMC Biology 05/2010; 8(1). DOI:10.1186/1741-7007-8-57 · 7.98 Impact Factor
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    • "The protein has been circularly permutated (Koebink and Kramer 1995) (in this case this is where the N-and C-termini have been swapped from the periplasmic side to the exterior of the outer membrane) so that protein tags can be fused to the OmpA scaVold whilst still allowing for surface immobilisation. OmpA is immobilised to gold surfaces through a cysteine residue that has been inserted into the periplasmic turn four (Shah et al. 2007). The circular permutation, beta-barrel structure and gold immobilisation make the OmpA an ideal scaVold protein for use in membrane arrays since protein domains can be genetically fused to its N-or C-terminus. "
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    ABSTRACT: Protein arrays are used in a wide range of applications. The array described here binds IgG antibodies, produced in rabbit, to gold surfaces via a scaffold protein. The scaffold protein is a fusion of the monomeric E. coli porin outer membrane protein A (OmpA) and the Z domain of Staphylococcus aureus protein A. The OmpA binds to gold surfaces via a cysteine residue in a periplasmic turn and the Z domain binds immunoglobulins via their constant region. Polarised Neutron Reflection is used to probe the structure perpendicular to the gold surface at each stage of the assembly of the arrays. Polarised neutrons are used as this provides a means of achieving extra contrast in samples having a magnetic metal layer under the gold surface. This contrast is attained without resorting to hydrogen/deuterium exchange in the biological layer. Polarised Neutron Reflection allows for the modelling of many and complex layers with good fits. The total thickness of the biological layer immobilised on the gold surface is found to be 187 A and the layer can thus far be separated into its lipid, protein and solvent parts.
    European Biophysics Journal 07/2008; 37(5):639-45. DOI:10.1007/s00249-008-0291-2 · 2.22 Impact Factor
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