Zooming in: Structural Investigations of Rheologically Characterized Hydrogen-Bonded Low-Methoxyl Pectin Networks

Self-assembled hydrogen-bonded networks of the polysaccharide pectin, a mechanically functional component of plant cell walls, have been of recent interest as biomimetic exemplars of physical gels, and the microrheological and strain-stiffening behaviors have been previously investigated. Despite this detailed rheological characterization of preformed gels, little is known about the fundamental arrangement of the polymers into cross-linking junction zones, the size of these bonded regions, and the resultant network architecture in these hydrogen-bonded materials, especially in contrast to the plethora of such information available for their well-known calcium-assembled counterparts. In this work, in concert with pertinent rheological measurements, an in-depth structural study of the hydrogen-bond-mediated gelation of pectins is provided. Gels were realized by using glucona-delta-lactone to decrease the pH of solutions of pectic polymers that had a (blockwise) low degree of methylesterification. Small-angle X-ray scattering and transmission electron microscopy were utilized to access structural information on length scales on the order of nanometers to hundreds of nanometers, while complementary mechanical properties were measured predominantly using small amplitude oscillatory shear rheology

Oligomeric Gag has increased binding preference towards A-containing over GU-containing RNA sequences.

<p><b>(A)</b> Calculated binding affinities (<i>K</i>_<i>d</i>) from ITC binding curves fitted to a one-site interaction model are plotted for replicate interactions of first set of GU-containing and A-containing RNA (4x 5’-GUAGG-3’ and 4x 5’-GAGAA-3’, respectively) and second set of GU-containing and A-containing RNA (4x 5’-UGUGG-3’ and 4x 5’-AAGGA-3’, respectively) with Pr55^Gag, Pr50^GagΔp6, Pr55^GagWM, p15^NC-SP2-p6 and p7^NC. <b>(B)</b> Calculated binding affinities (<i>K</i>_<i>d</i>) for Pr55^Gag and Pr55^GagWM for replicate sets of interactions were tabulated and the fold difference between GU-containing and A-containing RNA binding affinities for each set were calculated. The average fold difference across the 4 sets is highlighted in bold. Sets 1 and 2 represent <i>K</i>_<i>d</i> values obtained from replicate Gag interactions with 4x 5’-GUAGG-3’ (GU-containing RNA) and 4x 5’-GAGAA-3’ (A-containing RNA). Sets 3 and 4 represent <i>K</i>_<i>d</i> values obtained from replicate Gag interactions with a second set of GU-containing and A-containing RNA (4x 5’-UGUGG-3’ and 4x 5’-AAGGA-3’, respectively). <b>(C)</b> Proposed model for Pr55^Gag trafficking and viral RNA interaction during viral assembly. The schematic representation of GU-containing and A-containing RNA motifs aims to represent how Gag interacts with different RNA motifs during virus biogenesis. The distributions of these RNA motifs are not evenly spread-out across HIV RNA genome, and more precise distributions of these RNA can be seen in <b><a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1006221#ppat.1006221.s003" target="_blank">S3 Fig</a></b>.</p

Binding between Psi SL3 RNA and Pr55^Gag is an energetically favourable reaction and the level of energy released is enhanced by the oligomerization capacity of Pr55^Gag.

<p><b>(A)</b> Schematic representation of domain arrangement in Gag proteins (Pr55^Gag, Pr50^GagΔp6, Pr55^{Gag WM}) and processed NC proteins (p15^NC-SP2-p6 and p7^NC) used in this study <b>(B)</b> The top panels show the differential heat released following baseline subtraction and the bottom panels show representative ITC binding curve indicating heat released per mole of oligonucleotide titrated when Psi SL3 RNA (40 μM) was injected in 1.5 μL aliquots into 8 μM of Pr55^Gag, Pr50^GagΔp6, Pr55^{Gag WM}, p15^NC-SP2-p6 or p7^NC, respectively. Calculated <b>(C)</b> binding enthalpies (ΔH), <b>(D)</b> binding entropies (minus TΔS), <b>(E)</b> Gibb’s free energies (ΔG) and <b>(F)</b> binding affinities (<i>k</i>_d) from ITC binding curves fitted to a one-site interaction model plotted for replicate Gag protein interactions with Psi SL3 RNA (n = 3). The mean value is plotted and the error bars represent the SEM.</p

ITC analysis of Gag-RNA binding show more energetically favourable binding enthalpies and larger unfavourable entropy of Gag to A-containing over GU-containing RNA.

<p>Calculated <b>(A)</b> binding enthalpy, <b>(B)</b> binding entropy and <b>(E)</b> Gibb’s Free Energy from ITC binding curves fitted to a one-site interaction model plotted for replicate Gag protein interactions with first set of 20mers GU-containing RNA (4x 5’-GAUGG-3’) and A-containing RNA (4x 5’-GAGAA-3’) (n = 3). Calculated <b>(C)</b> binding enthalpy and <b>(D)</b> binding entropy and <b>(F)</b> Gibb’s Free Energy from ITC binding curves with second set of 20mers GU-containing (4x 5’-UGUGG-3’) and A-containing RNA (4x 5’-AAGGA-3’) (n = 2). The mean value is plotted and the error bars represent the SEM.</p

Stoichiometry of binding indicates higher number of Gag binds to A-containing RNA over GU-containing RNA and SL3.

<p>Calculated binding stoichiometry (N) from ITC binding curves fitted to a one-site interaction model plotted for replicate Gag protein interactions with SL3 RNA, GU-containing RNA (4’x 5’-GAUGG-3’) and A-containing RNA (4x 5’-GAGAA-3’) (n = 3). The binding stoichiometry (N) refers to RNA:Gag ratio.</p

Favourable interaction energetics between high-order Pr55^Gag oligomers and Adenosine-containing RNA persists with authentic HIV A-containing sequences.

<p><b>(A)</b> On the left is a top-down view of the published immature Pr55^Gag capsid hexamer cryo-EM structure (PDB ID:4usn) [<a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1006221#ppat.1006221.ref032" target="_blank">32</a>]. Intra-hexameric subunits are coloured in alternating green-cyan and dark green. Helix6 mutations (TTSTLQ 239–44 AASALA) disrupting intra-hexameric interactions of the NTD-capsid are shown as spheres and coloured in magenta. Helix10 mutation (D329A) and MHR mutation (K290A) disrupting intra-hexameric interactions of the CTD-capsid are shown as spheres and coloured in orange and blue-white, respectively. On the right is the side view of immature Pr55^Gag capsid. The WM mutation (WM316-7AA) disrupting inter-hexameric dimerization is highlighted in blue. Pr55^Gag (CA All 4) contains 4 sets of mutations that disrupt CA oligomerization (Helix6, Helix10, WM and MHR) [<a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1006221#ppat.1006221.ref028" target="_blank">28</a>]. ITC binding curves indicating heat released per mole of oligonucleotide titrated when 40 μM of 4x 5’-GAUGG-3’ (GU-containing) and 4x 5’-GAGAA-3’ (A-containing) RNA were injected in 1.5 ul aliquots into 8 μM of Pr55^Gag (CA Helix 6) (Fig 6A left panel, dark pink and light pink), Pr55^Gag (CA Helix 10) (Fig 6A middle panel, dark yellow and light yellow), and Pr55^Gag (CA All 4) (Fig 6A right panel, red-brown and purple). (n = 3) <b>(B)</b> Representative ITC binding curves indicating heat released per mole of oligonucleotide titrated when 40 μM of 4x 5’-GAUGG-3’ (GU-containing) and 4x 5’-GAGAA-3’ (A-containing) RNA were injected in 1.5 ul aliquots into 8 μM of Pr55^Gag-Tev (Fig 6B left panel, dark blue and light blue), Pr50^{Gag Δp6-TEV} (Fig 6B middle panel, dark orange and light orange), and Pr55^{Gag WM-TEV} (Fig 6B right panel, green and dark yellow). (n = 3) <b>(C)</b> Representative ITC binding curves indicating heat released per mole of oligonucleotide titrated when 40 μM of 5’- CTTAGAAATAGGGCAGCATAGAACAAAAATAGAGGAA-3’ (HIV_NL4.3RNA_2671-2710) and 5’- CTATCTTTTAGGGCAGCAATCTTCAAAAATAGTCCTT-3’ (HIV_NL4.3RNA_2671-2710 with AGAAA mutation) RNA were injected in 1.5 ul aliquots into 8 μM of Pr55^Gag-Tev (Fig 6C left panel, dark blue and light blue), Pr50^{Gag Δp6-TEV} (Fig 6C middle panel, dark orange and light orange), and Pr55^{Gag WM-TEV} (Fig 6C right panel, green and dark yellow). (n = 3)</p

Protein composition of <i>Gemmata obscuriglobus</i> pore-containing membrane.

<p><b>(<i>A</i>)</b> SDS-PAGE gel showing that <i>G</i>.<i>obscuriglobus</i> cells have three different types of membranes. Exclusively pore-containing membranes (fraction 3) display a characteristic protein profile distinct from that of membrane fractions which do not possess pore structures. <b>(<i>B</i>)</b> Venn diagram showing the number and distribution of proteins among the fractions and (in brackets) the number of proteins with the beta-propeller folds. The members of the beta-propeller cluster belong either exclusively to fraction 3 (4 proteins), or to fractions 3 and 2 (2 proteins), and to fractions 2, 3 and 6 (2 proteins). No beta-propeller containing proteins were found exclusively in fractions 2 or 6. <b>(<i>C</i>)</b> A beta-propeller family found in fraction 3 (pore-containing membranes), including some exclusive to fraction 3. Cluster analyses revealed a set of proteins with conserved C-terminal regions (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0169432#pone.0169432.s017" target="_blank">S13</a>–<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0169432#pone.0169432.s020" target="_blank">S16</a> Figs) that model beta-propeller folds with high (>95%) confidence. Models 3 (for protein ZP_02737072), 4 (ZP_02736670), 5 (ZP_02734776) and 6 (ZP_ZP_02734577) were deduced from proteins found exclusively in fraction 3 (pore-containing fraction); models 2 (for ZP_02737073) and 7 (for ZP_02733245) were deduced from proteins found in fractions 3 and 2 only; models 1 (for ZP_02737797) and 8 (for ZP_02731113)–for proteins found in fractions 3, 2, and 6 (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0169432#pone.0169432.s027" target="_blank">S4 Table</a>).</p

Pores in the membranes of <i>Gemmata obscuriglobus</i> released via sonication.

<p><b>(<i>A</i></b>) Transmission electron micrograph of a membrane fragment released from a lysed cell via sonication and negatively stained with ammonium molybdate. Large pores (arrows) with relatively electron-dense pore centers surrounded by a thin lighter inner ring and a thicker outer ring are seen. Smaller pore structures (arrowheads) are also visible and may represent either another class of pores or a result of a reverse view of the same large pores resulting from overlapping folds in the membrane (evidence for such structures is not derived from other microscopy methods). Bar, 100 nm. <b>Inset:</b> enlargement of boxed large pore in main Fig where a pore centre (PC), an inner ring (IR) and an outer ring (OR) can be distinguished. Bar, 50 nm. (<b><i>B</i></b>) TEM of a pore seen in negatively stained membrane fraction isolated from sonication-lysed cells, showing pore complex structure including outer ring (OR), inner ring (IR), spokes connecting inner and outer rings (S) and central plug (CP). Bar, 30 nm. <b>(<i>C</i>)</b> Enlarged view of the inner ring (IR) and central plug (CP) of the boxed pores in Fig 3A, the octagonal shape of the rings (especially visible if the outer edge of the outer ring is traced) is consistent with an eight-fold symmetry. Bar, 15 nm.</p

<i>Gemmata obscuriglobus</i> internal membrane pores as seen in freeze-fractured cells.

<p><b>(<i>A</i>)</b> Transmission electron micrograph of a platinum/carbon (Pt/C)-shadowed replica of a whole cell of <i>G</i>. <i>obscuriglobus</i> which has been prepared via the freeze-fracture technique. Bar, 100 nm. Inside the cell, a large spherical internal organelle consistent with the nuclear body organelle surrounding the nucleoid has been fractured (split) along and through the surface membranes of its envelope. Pores with a central core and at least one surrounding ring are visible on one region of one of the membranes of this organelle surface. Insets represent successive enlarged views of the boxed region in the main image displaying the pores at higher magnification. Bars, 100nm. At the highest enlargement the substructure of each of several pores can be resolved including central core and surrounding inner dark and outer light rings (right inset). (<b><i>B</i></b>) This micrograph of the whole cell reveals an apparently cross-fractured major internal organelle compartment and a membrane surface (boxed) representing a fracture through the membrane surrounding the organelle. Bar, 200 nm. <b>(<i>C</i>)</b> An enlarged view of the boxed region of the freeze-fractured cell seen in Fig 2B showing a region of a membrane surface where roughly circular pore structures (arrowheads) are visible, in some cases with two light rings surrounding a dark centre,. Bar, 50 nm.</p

Model of the pore complex of <i>Gemmata obscuriglobus</i>.

<p>The pore complex is composed of at least two concentric upper rings (blue), and a lower ring (light blue) connected by struts to a distal ring (green) to form a basket structure. The central plug (purple) rests within the inner ring and spans the length of the pore. The whole pore complex rests within membrane (orange). The structure and dimensions are based on available data from all EM methods applied, from both whole cells and fraction 3 isolated membranes, and with minimal extrapolation, so that although the pore is probably not a hollow structure the space within the pore has not been filled in.</p