Biomod/2013/Harvard/results

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==Input==
==Input==
One round of directed evolution produced one library member with improved affinity to δ-toxin. Variant H3 saw a marked increase in δ-toxin activity over the negative control (no peptide). While the improved activity is not nearly as high as the m13 activity, this variant appears to have begun mutating in the correct direction to improve binding. For variant H3, activity with both m13 and δ-toxin has increased, suggesting that any improvements to δ-toxin binding are not specific to δ-toxin. This evolution of "promiscuity," where a protein expands the number of targets it can bind to, is seen as a crucial intermediate step in directed evolution of novel protein targets ([[Biomod/2013/Harvard/References#Input_Domain |Bloom JD, Arnold FH. 2009. <i>PNAS</i>. 106 (Supplement 1) 9995-10000]]).
One round of directed evolution produced one library member with improved affinity to δ-toxin. Variant H3 saw a marked increase in δ-toxin activity over the negative control (no peptide). While the improved activity is not nearly as high as the m13 activity, this variant appears to have begun mutating in the correct direction to improve binding. For variant H3, activity with both m13 and δ-toxin has increased, suggesting that any improvements to δ-toxin binding are not specific to δ-toxin. This evolution of "promiscuity," where a protein expands the number of targets it can bind to, is seen as a crucial intermediate step in directed evolution of novel protein targets ([[Biomod/2013/Harvard/References#Input_Domain |Bloom JD, Arnold FH. 2009. <i>PNAS</i>. 106 (Supplement 1) 9995-10000]]).
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[[Image:Inputresultsgraph.png|frame|center|400px|Library Member H3 displayed an improvement in δ-toxin activity above the negative control]]
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[[Image:Inputresultsgraph.png|frame|center|250px|Library Member H3 displayed an improvement in δ-toxin activity above the negative control]]
==Output==
==Output==

Revision as of 12:36, 24 October 2013

Results

Contents


Input

One round of directed evolution produced one library member with improved affinity to δ-toxin. Variant H3 saw a marked increase in δ-toxin activity over the negative control (no peptide). While the improved activity is not nearly as high as the m13 activity, this variant appears to have begun mutating in the correct direction to improve binding. For variant H3, activity with both m13 and δ-toxin has increased, suggesting that any improvements to δ-toxin binding are not specific to δ-toxin. This evolution of "promiscuity," where a protein expands the number of targets it can bind to, is seen as a crucial intermediate step in directed evolution of novel protein targets (Bloom JD, Arnold FH. 2009. PNAS. 106 (Supplement 1) 9995-10000).

Library Member H3 displayed an improvement in δ-toxin activity above the negative control
Library Member H3 displayed an improvement in δ-toxin activity above the negative control

Output

Cysteine Mutations

GLucCaM contains ten cysteine residues. Since our protein could not be deactivated with calcium, we considered the possibility of disulfide bonds forming across the protein, preventing conformation changes. Unfortunately, we do not know the specific amino acids responsible for the disulfide bonds, so we selected a reasonable set of four cysteine residues highlighted in yellow below. The light blue indicates the two halves of GLuc, the red indicates calmodulin, and the green regions indicates the two linkers.

Image:Mutation_Sites.png

Linker Variations

Another possibility we explored was varying the linker length and rigidity. The original GLucCam structure has amino acid strands GGGGT and GGGS as the first and second linker sequence. We explored the following linker variations:

Short-rigid: GPG GPG

Long-rigid: APAPAP APAPAP

Short-flexible: GGGGSGGGGS GGGGSGGGGS

Image:Linker_Results.png‎

All linker variations showed a decrease in luminescence level, but they all exhibited significant decrease in activity when exposed to high concentration of calcium. Unfortunately, the protein switch does not respond to increased levels of M13, but this is a promising start for continued modification to the output domain.

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