<h4style = "color:rgb(255, 255, 255)">Model of our engineered sequence is consistent with prior measurements of a disulfide bond</h4>
<p>
While the AlphaFold [1] predictions are low confidence of the h-fibroin structure as a whole, the use of energy minimization and molecular
While the AlphaFold [1] predictions are low confidence of the H-fibroin structure as a whole, the use of energy minimization and molecular
dynamics [2,3,4] builds confidence in the quality of this output. Most importantly, our model suggests that key residue-specific interactions
between l- and h-fibroin are preserved in our engineered sequences, relative to their native counterparts. A strong cysteine-cysteine disulfide
interaction between h- and l-fibroin is predicted to be preserved—for instance, the model suggests that the cysteine ~20 residues from the end
of our engineered h-fibroin and the cysteine between residues 150 and 200 of the l-fibroin are consistent with their expected relative positions
between L- and H-fibroin are preserved in our engineered sequences, relative to their native counterparts. A strong cysteine-cysteine disulfide
interaction between h- and L-fibroin is predicted to be preserved—for instance, the model suggests that the cysteine ~20 residues from the end
of our engineered H-fibroin and the cysteine between residues 150 and 200 of the L-fibroin are consistent with their expected relative positions
and that they maintain the strong cysteine-cysteine disulfide interaction observed in existing caddisfly silk proteins.[5]
</p>
<p>
In prior biochemical experiments on homologous l- and h-fibroin subunits in the silkworm Bombyx mori, performed by Tanaka et al., Cys-172 of
In prior biochemical experiments on homologous l- and H-fibroin subunits in the silkworm Bombyx mori, performed by Tanaka et al., Cys-172 of
L-chain and Cys-c20 of H-chain had been experimentally validated as the residues to form a disulfide bond in that species. These measurements and
validations guided our identification of similar interactions proposed by our model—between cysteine residues in the same regions.
</p>
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@@ -35,7 +35,7 @@
In short, Tanaka et al. identified the disulfide bond by chemical detection. They separated fractions of the whole peptide chains by chromatography,
which facilitated a search for the specific residues involved in disulfide bonding (divide and conquer). The fragments containing cysteine residues
were deduced by sequencing, and peptide sequence analysis indicated which fractions formed disulfide bonds. Two iterations of chromatography were enough
to deduce that h-fibroin Cys-c1 and Cys-c4 forms an intramolecular disulfide bond, while <b>Cys-c20 forms an intermolecular disulfide linkage with Cys-172
to deduce that H-fibroin Cys-c1 and Cys-c4 forms an intramolecular disulfide bond, while <b>Cys-c20 forms an intermolecular disulfide linkage with Cys-172
<figcaptionclass="figure-caption">Sequence of homologous h-fibroins and visualization of the beta-sheets (red, orange nodes). Note the high proportion of serine (S) residues. [8]</figcaption>
<figcaptionclass="figure-caption">Sequence of homologous H-fibroins and visualization of the beta-sheets (red, orange nodes). Note the high proportion of serine (S) residues. [8]</figcaption>
<h4style = "color:rgb(255, 255, 255)">Further refinements are achievable</h4>
<p>
The model can be improved through the use of Monte Carlo protein folding and docking as provided in software such as pyRosetta, and through finer refinement with full-atom simulations using
AMBER force fields. Neither of these tools was used for our preliminary simulations due to hardware constraints and software limitations; however, they are ideal next steps to more rigorously
develop these silk protein models. One current plan of model development involves the inclusion of divalent ions (Ca2+) in the simulation to assess how these synthetic caddisfly silk protein
structures may change as the interaction with ions is expected to cause the silk to harden into tough fibers [6].
