EXACTIN-A SPECIFIC INHIBTOR OF 'FACTOR X' ACTIVATION BY EXTRINSIC TENASE COMPLEX ISOLATED FROM HEMACHATUS HAEMACHATUS VENOM

Ph.DDOCTOR OF PHILOSOPH

Identification and Structural Characterization of a New Three-Finger Toxin Hemachatoxin from Hemachatus haemachatus Venom

10.1371/journal.pone.0048112PLoS ONE710

Nanoencapsulation as a General Solution for Lyophilization of Labile Substrates

Protein macromolecules occur naturally at the nanoscale. The use of a dedicated nanoparticle as a lyophilization excipient, however, has not been reported. Because biopolymeric and lipid nanoparticles often denature protein macromolecules and commonly lack the structural rigidity to survive the freeze-drying process, we hypothesized that surrounding an individual protein substrate with a nanoscale, thermostable exoshell (tES) would prevent aggregation and protect the substrate from denaturation during freezing, sublimation, and storage. We systematically investigated the properties of tES, including secondary structure and its homogeneity, throughout the process of lyophilization and found that tES have a near 100% recovery following aqueous reconstitution. We then tested the hypothesis that tES could encapsulate a model substrate, horseradish peroxidase (HRP), using charge complementation and pH-mediated controlled assembly. HRP were encapsulated within the 8 nm internal tES aqueous cavity using a simplified loading procedure. Time-course experiments demonstrated that unprotected HRP loses 95% of activity after 1 month of lyophilized storage. After encapsulation within tES nanoparticles, 70% of HRP activity was recovered, representing a 14-fold improvement and this effect was reproducible across a range of storage temperatures. To our knowledge, these results represent the first reported use of nanoparticle encapsulation to stabilize a functional macromolecule during lyophilization. Thermostable nanoencapsulation may be a useful method for the long-term storage of labile proteins

Nanoencapsulation as a General Solution for Lyophilization of Labile Substrates

10.3390/pharmaceutics13111790PHARMACEUTICS131

Thermostable exoshells fold and stabilize recombinant proteins

The expression and stabilization of recombinant proteins is fundamental to basic and applied biology. Here we have engineered a thermostable protein nanoparticle (tES) to improve both expression and stabilization of recombinant proteins using this technology. tES provides steric accommodation and charge complementation to green fluorescent protein (GFPuv), horseradish peroxidase (HRPc), and Renilla luciferase (rLuc), improving the yields of functional in vitro folding by ~100-fold. Encapsulated enzymes retain the ability to metabolize small-molecule substrates, presumably via four 4.5-nm pores present in the tES shell. GFPuv exhibits no spectral shifts in fluorescence compared to a nonencapsulated control. Thermolabile proteins internalized by tES are resistant to thermal, organic, chaotropic, and proteolytic denaturation and can be released from the tES assembly with mild pH titration followed by proteolysis.NMRC (Natl Medical Research Council, S’pore)Published versio

Purification of hemachatoxin from the venom of <i>H. haemachatus</i>.

<p>(<b>A</b>) Size-exclusion chromatogram of the crude venom. The proteins were eluted using 50 mM Tris-HCl, pH 7.4 and monitored at 280 nm. The fractions of peak 3 (<i>black horizontal bar</i>) were pooled and sub-fractionated on RP-HPLC. (<b>B</b>) RP-HPLC chromatogram of peak 3 using a linear gradient of 28–50% solvent B. The elution was monitored at 215 nm. The <i>black arrow</i> indicates the elution of hemachatoxin. (<b>C</b>) The re-purification of hemachatoxin on a shallow gradient of 35–45% solvent B. The elution was monitored at 215 nm. (<b>D</b>) The ESI-MS profile of hemachatoxin showing the three peaks of mass/charge (m/z) ratio ranging from +4 to +6 charges. The mass of hemachatoxin was determined to be 6835.68±0.94 Da.</p

Crystallographic data and refinement statistics.

<p>Statistics from the current model.</p>a<p>R_sym = Σ|I_i−<i>|/Σ|I_i| where I_i is the intensity of the i^th measurement, and <i> is the mean intensity for that reflection.</i></i></p><i><i>b<p>R_work = Σ| F_obs−F_calc|/Σ|F_obs| where F_calc and F_obs are the calculated and observed structure factor amplitudes, respectively.</p>c<p>R_free = as for R_work, but for 10.0% of the total reflections chosen at random and omitted from refinement.</p>*<p>Values in the parenthesis are the highest resolution bin values.</p></i></i

A general approach to protein folding using thermostable exoshells

10.1038/s41467-021-25996-4NATURE COMMUNICATIONS12

Comparison of hemachatoxin with other three-finger toxins.

<p>(<b>A</b>) Structure based sequence alignment of hemachatoxin and its homologs, cardiotoxin 3 (1H0J), cytotoxin 3 (1XT3), cardiotoxin A3 (2BHI), cardiotoxin VI (1UG4) and cardiotoxin V (1KXI), (all from <i>Naja atra</i>), cardiotoxin V_II4 (1CDT) from <i>Naja mossambica</i> and toxin-γ (1TGX) (a cardiotoxin from <i>Naja nigricollis</i>). This figure was generated using the programs ClustalW <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0048112#pone.0048112-Larkin1" target="_blank">[78]</a> and ESPript <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0048112#pone.0048112-Gouet1" target="_blank">[79]</a>. (<b>B</b>) Comparison of hemachatoxin with its structural homologs. Hemachatoxin (brown), cardiotoxin 3 [1H0J] (cyan), cytotoxin 3 [1XT3] (black), carditotoxin A3 [2BHI] (blue), cardiotoxin VI [1UG4] (red), cardiototoxin V [1KXI] (pink), cardiotoxin V_II4 [1CDT] (green) and toxin-γ [1TGX] (yellow).</p

Multiple sequence alignment of hemachatoxin with cardiotoxins/cytotoxins (A) and other three-finger toxins (B).

<p>Toxin names, species and accession numbers are shown. Conserved residues in all the sequences are highlighted in black. The type of cardiotoxin based on the conserved Pro31 is highlighted in grey. Disulfide linkages and loop regions are also shown. The sequence identity (in percentage) of each protein with hemachatoxin is shown at the end of each sequence.</p