Investigation of lsm proteins as scaffolds in bionanotechnology

Self-assembling materials have gained attention in the field of nanotechnology due to their potential to be used as building blocks for fabricating complex nanoscale devices. The biological world is abundant with examples of functional self-assembling biomolecules. Proteins are one such example, found in a variety of geometries and shapes. This research is focussed on the use of ring-shaped self-assembling proteins, called Lsm proteins, as componentary for applications in bionanotechnology. Lsm proteins were used because of their spontaneous association into stable rings, tolerance to mutations, and affinity to RNA. This thesis primarily focussed on the thermophilic Lsmα (from Methanobacterium. thermoautotrophicum) that assembles as heptameric rings. The oligomeric state of the heptameric protein, and hence the diameter of its central cavity, was manipulated by judiciously altering appropriate residues at the subunit interface. Lsmα presented a complex set of interactions at the interface. Out of the mutations introduced, R65P yielded a protein for which SEC and SAXS data were consistent with a hexameric state. Moreover, key residues, L70 and I71, were identified that contribute to the stability of the toroid structure. Covalent linking of rings provided nanotubular structures. To achieve this, the surface of the Lsmα ring scaffold was modified with Cys residues. This approach led to the formation of novel Lsmα nanotubes approximately 20 nm in length. Importantly, the assembly could be controlled by changing the redox conditions. As an alternative method to manipulate the supramolecular assembly, His6-tags were attached at the termini of the Lsmα sequence. The higher-order organisation of the constructs was influenced by the position of the His6-tag. The N-terminally attached His6-tag version of Lsmα showed a metal-dependent assembly into cage-like structures, approximately 9 nm across. This organisation was highly stable, reproducible, and reversible in nature. The results presented in this thesis aid the understanding of generating complex nanostructures via in vitro self-assembly. The Lsmα rings were assembled into higher-order architectures at the quaternary level by employing protein engineering strategies. Future work is necessary to functionalise these supramolecular structures; however, this study confirms the potential role of Lsmα proteins as a molecular building block in bionanotechnology

Protein nanorings organized by poly(styrene-block-ethylene oxide) self-assembled thin films

This study explores the use of block copolymer self-assembly to organize Lsmα, a protein which forms stable doughnut-shaped heptameric structures. Here, we have explored the idea that 2-D crystalline arrays of protein filaments can be prepared by stacking doughnut shaped Lsmα protein into the poly(ethylene oxide) blocks of a hexagonal microphase-separated polystyrene-b-polyethylene oxide (PS-b-PEO) block copolymer. We were able to demonstrate the coordinated assembly of such a complex hierarchical nanostructure. The key to success was the choice of solvent systems and protein functionalization that achieved sufficient compatibility whilst still promoting assembly. Unambiguous characterisation of these structures is difficult; however AFM and TEM measurements confirmed that the protein was sequestered into the PEO blocks. The use of a protein that assembles into stackable doughnuts offers the possibility of assembling nanoscale optical, magnetic and electronic structures

Characterisation of the First Enzymes Committed to Lysine Biosynthesis in <em>Arabidopsis thaliana</em>

<div><p>In plants, the lysine biosynthetic pathway is an attractive target for both the development of herbicides and increasing the nutritional value of crops given that lysine is a limiting amino acid in cereals. Dihydrodipicolinate synthase (DHDPS) and dihydrodipicolinate reductase (DHDPR) catalyse the first two committed steps of lysine biosynthesis. Here, we carry out for the first time a comprehensive characterisation of the structure and activity of both DHDPS and DHDPR from <em>Arabidopsis thaliana</em>. The <em>A. thaliana</em> DHDPS enzyme (<em>At</em>-DHDPS2) has similar activity to the bacterial form of the enzyme, but is more strongly allosterically inhibited by (<em>S</em>)-lysine. Structural studies of <em>At</em>-DHDPS2 show (<em>S</em>)-lysine bound at a cleft between two monomers, highlighting the allosteric site; however, unlike previous studies, binding is not accompanied by conformational changes, suggesting that binding may cause changes in protein dynamics rather than large conformation changes. DHDPR from <em>A. thaliana</em> (<em>At</em>-DHDPR2) has similar specificity for both NADH and NADPH during catalysis, and has tighter binding of substrate than has previously been reported. While all known bacterial DHDPR enzymes have a tetrameric structure, analytical ultracentrifugation, and scattering data unequivocally show that <em>At</em>-DHDPR2 exists as a dimer in solution. The exact arrangement of the dimeric protein is as yet unknown, but <em>ab initio</em> modelling of x-ray scattering data is consistent with an elongated structure in solution, which does not correspond to any of the possible dimeric pairings observed in the X-ray crystal structure of DHDPR from other organisms. This increased knowledge of the structure and function of plant lysine biosynthetic enzymes will aid future work aimed at improving primary production.</p> </div

Analytical ultracentrifugation of <i>At</i>-DHDPS2 and <i>At</i>-DHDPR2.

<p>Sedimentation velocity analysis of <i>At</i>-DHDPS2 and <i>At</i>-DHDPR2. A) Continuous sedimentation coefficient distribution [<i>(c)s</i>] analysis of <i>At-</i>DHDPS2 at a concentration of 0.75 mg.mL⁻¹ (black line). The RMSD and Runs Test Z (RTZ) scores for the fit were 0.008 and 3.2 respectively. B) <i>(c)s</i> analysis of <i>At-</i>DHDPR2 at concentrations of 0.1 mg.mL⁻¹ (black line; RMSD = 0.009, RTZ = 2.4), 0.2 mg.mL⁻¹ (red line; RMSD = 0.010, RTZ = 2.0), 0.4 mg.mL⁻¹ (green line; RMSD = 0.014, RTZ = 8.6) 0.8 mg.mL⁻¹ (pink line; RMSD = 0.013, RTZ = 4.9) and 1.6 mg.mL⁻¹ (blue line; RMSD = 0.015, RTZ = 7.4). Radial absorbance data for the three lower protein concentrations were acquired at a different wavelength to those of the two highest protein concentrations, and the <i>c(s)</i> distributions were scaled accordingly. Residuals for the fits are shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0040318#pone.0040318.s007" target="_blank">Figure S7</a>.</p

X-Ray scattering of DHDPR.

<p>Data were collected for <i>At</i>-DHDPR2, <i>Ec</i>-DHDPR and <i>Tm</i>-DHDPR (panel A); curves have been arbitrarily displaced along the logarithmic axis for clarity. Data was analysed using GNOM (fitted data shown by red line in panel A) to calculate a distance distribution function for each enzyme (panel B).</p

Analysis of DHDPR interface regions using the PDBePISA web server (<i>38</i>).

<p>Analysis of DHDPR interface regions using the PDBePISA web server (<i>38</i>).</p

Kinetic parameters (± SE) for <i>At</i>-DHDPR2 were calculated as described in materials and method section.

<p>Kinetic parameters (± SE) for <i>At</i>-DHDPR2 were calculated as described in materials and method section.</p

Crystal structures of unliganded and lysine bound <i>At</i>-DHDPS2.

<p>A) Wall-eyed stereo image of the Cα superposition of <i>At-</i>DHDPS2 with bound lysine (blue Cα trace) and unliganded <i>At-</i>DHDPS2 (gold Cα trace; rmsd = 0.3 Å). The lysine molecules bound at the allosteric site of each monomer of the tetramer are shown in yellow (stick representation). B) The lysine binding site at the monomer-monomer interface of the tight-dimer showing residues in contact with the bound lysine molecules (yellow). Electron density around the bound lysine (grey mesh, contoured at 1.0 sigma) was calculated using refined coordinates omitting the bound lysine molecules. Residues contributed by each monomer of the tight-dimer are shown in different shades of blue, and are indicated by the use of the prime (’) symbol. C) overlay of the lysine binding residues of the tight-dimer from the lysine bound (blue) and unliganded (gold) structures. Lysine molecules are shown in yellow. Residues contributed by each monomer of the tight-dimer are shown in different shades of blue or gold, and are indicated by the use of the prime (’) symbol.</p