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<div class="main ui citing justified container"> | <div class="main ui citing justified container"> | ||
− | <h1 class="ui left dividing header"><span id="intro" class="section"> </span>Coiled | + | <h1 class="ui left dividing header"><span id="intro" class="section"> </span>Coiled-coil interaction model</h1> |
<div class="ui segment"> | <div class="ui segment"> | ||
<p>Logic operations in biological systems have been tested with several approaches | <p>Logic operations in biological systems have been tested with several approaches | ||
<x-ref>Singh2014</x-ref> | <x-ref>Singh2014</x-ref> | ||
. Our project | . Our project | ||
− | relies on the reconstitution of split protein promoted by coiled coil (CC) dimerization. The | + | relies on the reconstitution of split protein promoted by coiled-coil (CC) dimerization. The |
interaction between CC peptides can be finely tuned | interaction between CC peptides can be finely tuned | ||
<x-ref>Woolfson2005, Gradisar2011, Negron2014</x-ref> | <x-ref>Woolfson2005, Gradisar2011, Negron2014</x-ref> | ||
, thereby CCs offers a flexible and | , thereby CCs offers a flexible and | ||
versatile platform in terms of designing logic operation <i>in vivo</i>. With the purpose of | versatile platform in terms of designing logic operation <i>in vivo</i>. With the purpose of | ||
− | understanding the relation that underlies the interaction between coiled coil peptides and | + | understanding the relation that underlies the interaction between coiled-coil peptides and |
therefore using them in logic gates, we designed the following model ( | therefore using them in logic gates, we designed the following model ( | ||
<ref>5.4.1.</ref> | <ref>5.4.1.</ref> | ||
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</div> | </div> | ||
− | <p>This relationship suggested to try using a different version of the coiled coils available in | + | <p>This relationship suggested to try using a different version of the coiled-coils available in |
the | the | ||
toolset already used by the <a href="https://2009.igem.org/Team:Slovenia">Slovenian iGEM 2009 | toolset already used by the <a href="https://2009.igem.org/Team:Slovenia">Slovenian iGEM 2009 | ||
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in | in | ||
vivo </a> we decided | vivo </a> we decided | ||
− | to use an inhibitory coiled coil, which would be displaced by the second coiled coil with | + | to use an inhibitory coiled-coil, which would be displaced by the second coiled-coil with |
higher | higher | ||
affinity, only once is cleaved off its partner ($ Kd_B \lt Kd_b $). In doing so we selected | affinity, only once is cleaved off its partner ($ Kd_B \lt Kd_b $). In doing so we selected | ||
P3 as | P3 as | ||
<b>B</b> and | <b>B</b> and | ||
− | P3mS as <b>b</b>, these two coiled coil peptides present only few substitutions and the higher | + | P3mS as <b>b</b>, these two coiled-coil peptides present only few substitutions and the higher |
solubility of P3mS (<b>b</b>), which presents Gln and Ser instead of Ala in <i>b</i> and <i>c</i> position of the | solubility of P3mS (<b>b</b>), which presents Gln and Ser instead of Ala in <i>b</i> and <i>c</i> position of the | ||
heptads, would favour the dissociation. We also tried differently destabilized versions of | heptads, would favour the dissociation. We also tried differently destabilized versions of | ||
Line 209: | Line 209: | ||
(obtained by substituting a and d positions with Ala) leads to a small difference of the | (obtained by substituting a and d positions with Ala) leads to a small difference of the | ||
signal | signal | ||
− | before and after cleavage. Using a slightly destabilized coiled coil (P3mS-2A), which | + | before and after cleavage. Using a slightly destabilized coiled-coil (P3mS-2A), which |
presents | presents | ||
only 2 alanines in the second heptad, the signal after cleavage reached its maximum of 16 | only 2 alanines in the second heptad, the signal after cleavage reached its maximum of 16 |
Revision as of 18:03, 18 October 2016
Coiled-coil interaction model
Logic operations in biological systems have been tested with several approaches
The relationship between the signal before and after cleavage by proteases is represented by the difference [AB] - [AB-b]. In order to understand the optimal combination of dissociation constant required to obtain a good signal we solved two systems of equations set up considering the two state of the reaction scheme, Before cleavage and After cleavage, (1) and (6) respectively, as separate phases of the reaction and additionally, considering cleavage as an irreversible and complete reaction.
Given values for total concentrations and Kd (from 10-9 to 10-3 M) the equations, for the reaction constants (2), (3) and (7), (8) and and for mass conservation (4), (5) and (9), (10), (11) were solved for the species at equilibrium.
Before cleavage \begin{equation} \ce{Axb + B <=>[Kd_x] A-b + B <=>[Kd_B] AB-b} \end{equation} \begin{align} Kd_x &= \frac{[A-b]}{[Axb]} \label{1.1-2}\\ Kd_B &= \frac{[A-b] * [B]}{[AB - b]} \\ c_B &= [B] + [AB-b]\\ c_A-b &= [A-b]+[Axb]+[AB-b] \label{2.1-2} \end{align} After cleavage \begin{equation} \ce{Ab + B <=>[Kd_b] A + b + B <=>[Kd_B] AB + b} \end{equation} \begin{align} Kd_b &= \frac{[A] * [b]}{[Ab]} \label{1.3-4}\\ Kd_B &= \frac{[A] * [B]}{[AB]} \\ c_A &= [A]+[AB]+[Ab]\\ c_B &= [B] +[AB]\\ c_b &= [b] + [Ab] \label{2.3-5} \end{align} The two systems are connected by the relation between the dissociation constants $Kd_b$ and $Kd_x$, \begin{equation} Kd_x = Kd_b * 4 * 10^{-3} M^{-1} \end{equation} This relation approximates the higher affinity between the coils A and b when they are covalently linked by a short peptide (as in the system “Before cleavage”)The results have been plotted varying the $_Kd for the interaction of A with both B and b, against the difference [AB] - [AB-b], where [AB] is considered the signal after cleavage and [AB-b] the signal before cleavage (leakage). The system revealed that in order to obtain a high difference between signal and leakage a high affinity of the coil B for the coil A (low $Kd_B$) is required, while on the other hand an excessive destabilization of the autoinhibitory coil b (high $Kd_b$) would prevent the signal to be visible ( 5.4.2. ).
This relationship suggested to try using a different version of the coiled-coils available in
the
toolset already used by the Slovenian iGEM 2009
team