Difference between revisions of "Team:NTHU Formosa/Model"

 
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   <div class="w3-container w3-padding-24 w3-center">
 
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     <div class="w3-content"><br><br><br>
 
     <div class="w3-content"><br><br><br>
       <h2 class="w3-wide" style="font-size:60px;">Modeling</h2>
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       <h2 class="w3-center" style="font-size:60px; font-family:Quicksand;"><b>Modeling</b></h2>
  
  
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     <p class="w3-justify"><b><big><big>Motivation</big></big></b></p>
 
     <p class="w3-justify"><b><big><big>Motivation</big></big></b></p>
 
     <p class="w3-justify">
 
     <p class="w3-justify">
<br>    Our sensing module design relies on a special form of antibody, called nanobodies. Nanobodies are single variable domain antibody fragments (VHH) derived from heavy-chain-only antibodies in camelidae.
+
<br>    Our sensing module design relies on a special form of antibody, called nanobodies. Nanobodies are single variable domain antibody fragments (VHH) derived from heavy-chain-only antibodies in camelidae. Nanobodies are used to recognize wide varieties of ligands. Due to its unique features, such as high affinity and specificity for antigen, thermostability, nanoscale size, and soluble characteristic in aqueous solution, over two thousand nanobodies are available for recognizing different antigens including membrane-bound molecules and soluble molecules, including biomarkers, according to the information from iCAN database (Institute collection and Analysis of Nanobody).
  
<br><br>    Nanobodies are used to recognize wide varieties of ligands. Due to its unique features, such as high affinity and specificity for antigen, thermostability, nanoscale size, and soluble characteristic in aqueous solution, over two thousand nanobodies are available for recognizing different antigens including membrane-bound molecules and soluble molecules, including biomarkers, according to the information from iCAN database (Institute collection and Analysis of Nanobody).
+
<br><br>    Here we use nanobodies as the extracellular domain on our sensing module. We split the nanobodies into N-terminal and C-terminal fragments. Based on previous studies (Tang et al., 2013, 201), antigen binding induces the dimerization between N-terminal and C-terminal and thus triggers downstream gene expressions. Therefore, we tag our split nanobodies with a transmembrane domain on our sensing module along with other proteins. Theoretically, antigens will induce dimerization of these split sensing module and turn on downstream events.  
 
+
<br><br>    Here we use nanobodies as the extracellular domain on our sensing module. We split the nanobodies into N-terminal and C-terminal fragments. Based on previous studies (Tang et al., 2013, 201), antigen binding induces the dimerization between N-terminal and C-terminal and thus triggers downstream gene expressions. Therefore, we tag our split nanobodies with transmembrane domain on our sensing module along with other proteins. Theoretically, antigen will induce dimerization of these split sensing module and turn on downstream events.  
+
 
</p><br><br><br><br>
 
</p><br><br><br><br>
  
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     <p class="w3-justify">
 
     <p class="w3-justify">
  
<br>    To prove the concept, we use the most well-studied nanobodies, GFP, and its antigen, GFP binding protein, GBP. Since trigger of BioWatcher system depends on the dimerization of the split nanobodies as they approach and binds to the antigen, prior to any of our experiment, we use simulation to model our design and see if dimerization of GBP N and C terminals happens at the presence of GFP.
+
<br>    To prove the concept, we use the most well-studied nanobodies, GFP, and its antigen, GFP binding protein, GBP. Since trigger of BioWatcher system depends on the dimerization of the split nanobodies as they approach and binds to the antigen, prior to any of our experiment, we use simulation to model our design and see if dimerization of GBP N and C terminals happens at the presence of GFP. The GFP-GBP(N) complex were first aligned to reference (3OGO) to guarantee the possible binding interface, the simulation was focus on the binding interface of GBP(C) to GFP-GBP(N) complex.
 
+
<br><br>    The GFP-GBPN complex were first aligned to reference (3OGO) to guarantee the possible binding interface, the simulation was focus on the binding interface of GBPC to GFP-GBPN complex.
+
 
<p/><br><br><br><br>
 
<p/><br><br><br><br>
  
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     <p class="w3-justify">
 
     <p class="w3-justify">
  
<br>    To best describe the conformation of GBPC and GFP-GBPN complex in real cellular environment, OpenMM Python API is used for molecular dynamics simulation, and the suggested binding conformation is visualized by VMD (Visual Molecular Dynamics). </p><br><br><br><br>
+
<br>    To best describe the conformation of GBP(C) and GFP-GBP(N) complex in real cellular environment, OpenMM Python API is used for molecular dynamics simulation, and the suggested binding conformation is visualized by VMD (Visual Molecular Dynamics). </p><br><br>
  
 
       <p class="w3-justify"><b>Molecular Dynamics Simulation</b></p>
 
       <p class="w3-justify"><b>Molecular Dynamics Simulation</b></p>
 
       <p class="w3-justify">
 
       <p class="w3-justify">
  
<br>    Although the GFP-binding domains of GBPN and GBPC are well-studied structures, the GBPN and GBPC linkers have no homology model structures. Therefore, they were built directly from Discovery Studio as very long unstructured loops.  
+
   Although the GFP-binding domains of GBP(N) and GBP(C) are well-studied structures, the GBP(N) and GBP(C) linkers have no homology model structures. Therefore, they were built directly from Discovery Studio as very long unstructured loops.  
<br><br>    At the initial condition, the soluble folded parts of GFP and GBPN have been balanced in the prior 200ns simulation. A stable binding interface between them has been found. The whole simulation would perform 200ns to find a stable structure of GBPC binding interface.
+
<br><br>    At the initial condition, the soluble folded parts of GFP and GBP(N) have been balanced in the prior 200ns simulation. A stable binding interface between them has been found. The whole simulation would perform 200ns to find a stable structure of GBP(C) binding interface.
 
</p><br><br>
 
</p><br><br>
<br><br><br>
 
  
  
 
       <p class="w3-justify"><b>Molecular Dynamics Simulation Details</b></p>
 
       <p class="w3-justify"><b>Molecular Dynamics Simulation Details</b></p>
 
       <p class="w3-justify">
 
       <p class="w3-justify">
<br>
 
 
Package: OpenMM Python API<br>
 
Package: OpenMM Python API<br>
 
Forcefield: CHARMM36m with supplementary force objects from CHARMM-GUI<br>
 
Forcefield: CHARMM36m with supplementary force objects from CHARMM-GUI<br>
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Time step: 0.002 ps<br>
 
Time step: 0.002 ps<br>
 
NPT production run: 200 ns<br>
 
NPT production run: 200 ns<br>
</p><br><br><br><br>
+
</p><br><br>
  
 
       <p class="w3-justify"><b>Sequence of the system to be built</b></p>
 
       <p class="w3-justify"><b>Sequence of the system to be built</b></p>
 
       <p class="w3-justify">
 
       <p class="w3-justify">
<br>
 
 
GFP:<br>
 
GFP:<br>
 
VSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLKFICTTGKLPVPWPTLVTTLTYGVQC
 
VSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLKFICTTGKLPVPWPTLVTTLTYGVQC
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PNEKRDHMVLLEFVTAAGIT
 
PNEKRDHMVLLEFVTAAGIT
 
<br><br>
 
<br><br>
GBPN:<br>
+
GBP(N):<br>
 
DVQLQESGGGSVQAGEALRLSCVGSGYTSINPYMAWFRQAPGKEREGVAAISSGGQYTYYADSVKGRFTI
 
DVQLQESGGGSVQAGEALRLSCVGSGYTSINPYMAWFRQAPGKEREGVAAISSGGQYTYYADSVKGRFTI
 
SRDNAKNTMYLQMPSLKPDDSAKYYCAADFRRGGSWNVDPLRYDYQHWGQGTQVTVSS
 
SRDNAKNTMYLQMPSLKPDDSAKYYCAADFRRGGSWNVDPLRYDYQHWGQGTQVTVSS
 
<br><br>
 
<br><br>
GBPC:<br>
+
GBP(C):<br>
 
DVQLQESGGGSVQAGGSLRLSCAASGFPFSNYCMGWFRQAPGKEREGVATISRLGMFTEYADSVQGRFII
 
DVQLQESGGGSVQAGGSLRLSCAASGFPFSNYCMGWFRQAPGKEREGVATISRLGMFTEYADSVQGRFII
 
SRDNAQNMVFLQMNNLTPEDTAIYYCAAVSTSSSDCRPRLPSQEYTYWGQGTQVTVSSQ
 
SRDNAQNMVFLQMNNLTPEDTAIYYCAAVSTSSSDCRPRLPSQEYTYWGQGTQVTVSSQ
 
<br><br>
 
<br><br>
GBPN Linker:<br>
+
GBP(N) Linker:<br>
 
SAGGGGGSNAVGQDTQEVIVVP
 
SAGGGGGSNAVGQDTQEVIVVP
 
<br><br>
 
<br><br>
GPBC Linker:<br>
+
GBP(C) Linker:<br>
 
SAGGNAVGQDTQEVIVVP
 
SAGGNAVGQDTQEVIVVP
 
<br><br>
 
<br><br>
 
Helix: <br>
 
Helix: <br>
 
HSLPFKVVVISAILALVVLTIISLIILIMLWQKKPRYE
 
HSLPFKVVVISAILALVVLTIISLIILIMLWQKKPRYE
</p><br><br><br><br>
+
</p><br><br>
  
       <p class="w3-justify"><b>VMD(Visual Molecular Dynamics)</b></p>
+
       <p class="w3-justify"><b>VMD (Visual Molecular Dynamics)</b></p>
 
       <p class="w3-justify">
 
       <p class="w3-justify">
<br>    VMD is a computer program designed for visualizing the result of molecular dynamics simulations, especially for protein and lipid bilayer system. Also, VMD can
+
   VMD is a computer program designed for visualizing the result of molecular dynamics simulations, especially for protein and lipid bilayer system. Also, VMD can
 
animate and analyze the trajectory of a MD simulation then shows molecules in variety drawing methods and materials. So we used VMD to present the suggested binding conformation by a 3D model.
 
animate and analyze the trajectory of a MD simulation then shows molecules in variety drawing methods and materials. So we used VMD to present the suggested binding conformation by a 3D model.
 
</p><br><br><br><br>
 
</p><br><br><br><br>
  
     <p class="w3-justify"><b><big><big>Results&Conclusions</big></big></b></p>
+
     <p class="w3-justify"><b><big><big>Results & Conclusions</big></big></b></p>
 
     <p class="w3-justify">
 
     <p class="w3-justify">
  
<br>    After 200ns simulation, the suggested binding conformation of the complex is shown below.
 
  
<br><br><br>
+
<br>
 
+
 
 
+
     
 
+
   
+
        <video width="45%"  loop="true" align="right" autoplay="autoplay"  muted="true">
+
        <source type="video/mp4" src="https://static.igem.org/mediawiki/2018/b/bf/T--NTHU_Formosa--wgbpn.mp4"></source>
+
</video> 
+
        <video width="45%"  loop="true" align="left"  autoplay="autoplay"  muted="true">
+
        <source type="video/mp4" src="https://static.igem.org/mediawiki/2018/a/a0/T--NTHU_Formosa--wgbpc.mp4"></source>
+
</video> 
+
        <video width="45%"  loop="true" align="right" autoplay="autoplay"  muted="true">
+
        <source type="video/mp4" src="https://static.igem.org/mediawiki/2018/0/06/T--NTHU_Formosa--wgfp.mp4"></source>
+
 
</video>   
 
</video>   
           <video width="45%"  loop="true" align="left"  
+
           <video width="80%"  loop="true" align="center"  
 
       autoplay="autoplay"  muted="true">
 
       autoplay="autoplay"  muted="true">
 
         <source type="video/mp4" src="https://static.igem.org/mediawiki/2018/b/bb/T--NTHU_Formosa--w3.mp4"></source>
 
         <source type="video/mp4" src="https://static.igem.org/mediawiki/2018/b/bb/T--NTHU_Formosa--w3.mp4"></source>
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<br><br>    Here in the videos, we showed the N-terminal GBP and C-terminal GBP anchored on the cell membrane separately. At the presence of GFP, dimerization of the N-terminal and C-terminal GBP are formed when they are pulled together. Based on the results of the simulation that approve the design of our sensing module, we ran <a href="https://2018.igem.org/Team:NTHU_Formosa/Results">further experiments</a> and confirm the complete design for BioWatcher cells.  
+
<br><br>   After 200ns simulation, the suggested binding conformation of the complex is shown above. Here in the videos, we showed the N-terminal GBP (showed in yellow) and C-terminal GBP (showed in pink) anchored on the cell membrane separately. At the presence of GFP (showed in green), dimerization of the N-terminal and C-terminal GBP are formed when they are pulled together. Based on the results of the simulation that approve the design of our sensing module, we ran <a href="https://2018.igem.org/Team:NTHU_Formosa/Results">further experiments</a> and confirm the complete design for BioWatcher cells.  
 
</p><br><br><br><br>
 
</p><br><br><br><br>
  
            <p class="w3-justify" style="font-size:20px;"><b>Reference: </b></p><br>
+
    <p class="w3-justify"><b><big><big>Reference:</big></big></b></p>
            <p class="w3-justify" style="font-size:20px;">
+
    <p class="w3-justify" style="font-size:20px;">
1. Altintas, Z., and Tothill, I. (2013). Biomarkers and biosensors for the early diagnosis of lung cancer. Sensors Actuators, B Chem. 188, 988–998.<br>
+
1. Altintas, Z., and Tothill, I. (2013). Biomarkers and biosensors for the early diagnosis of lung cancer. Sensors Actuators, B Chem. 188, 988–998.<br><br><br>
  
 
   </div>
 
   </div>

Latest revision as of 15:37, 17 October 2018




Modeling


Motivation


  Our sensing module design relies on a special form of antibody, called nanobodies. Nanobodies are single variable domain antibody fragments (VHH) derived from heavy-chain-only antibodies in camelidae. Nanobodies are used to recognize wide varieties of ligands. Due to its unique features, such as high affinity and specificity for antigen, thermostability, nanoscale size, and soluble characteristic in aqueous solution, over two thousand nanobodies are available for recognizing different antigens including membrane-bound molecules and soluble molecules, including biomarkers, according to the information from iCAN database (Institute collection and Analysis of Nanobody).

  Here we use nanobodies as the extracellular domain on our sensing module. We split the nanobodies into N-terminal and C-terminal fragments. Based on previous studies (Tang et al., 2013, 201), antigen binding induces the dimerization between N-terminal and C-terminal and thus triggers downstream gene expressions. Therefore, we tag our split nanobodies with a transmembrane domain on our sensing module along with other proteins. Theoretically, antigens will induce dimerization of these split sensing module and turn on downstream events.





Overview


  To prove the concept, we use the most well-studied nanobodies, GFP, and its antigen, GFP binding protein, GBP. Since trigger of BioWatcher system depends on the dimerization of the split nanobodies as they approach and binds to the antigen, prior to any of our experiment, we use simulation to model our design and see if dimerization of GBP N and C terminals happens at the presence of GFP. The GFP-GBP(N) complex were first aligned to reference (3OGO) to guarantee the possible binding interface, the simulation was focus on the binding interface of GBP(C) to GFP-GBP(N) complex.





Methodology


  To best describe the conformation of GBP(C) and GFP-GBP(N) complex in real cellular environment, OpenMM Python API is used for molecular dynamics simulation, and the suggested binding conformation is visualized by VMD (Visual Molecular Dynamics).



Molecular Dynamics Simulation

  Although the GFP-binding domains of GBP(N) and GBP(C) are well-studied structures, the GBP(N) and GBP(C) linkers have no homology model structures. Therefore, they were built directly from Discovery Studio as very long unstructured loops.

  At the initial condition, the soluble folded parts of GFP and GBP(N) have been balanced in the prior 200ns simulation. A stable binding interface between them has been found. The whole simulation would perform 200ns to find a stable structure of GBP(C) binding interface.



Molecular Dynamics Simulation Details

Package: OpenMM Python API
Forcefield: CHARMM36m with supplementary force objects from CHARMM-GUI
Electrostatics: PME
VDW: 6-12 LJ with 1-4 scaling and force-switching
Switching: 1.0 nm
Cutoff: 1.2 nm
Integrator: LangevinIntegrator
Temperature: 310 K
Ion concentrations: 0.15 M NaCl
Barostat: MonteCarloMembraneBarostat
Volume changing frequency: 100 time steps
Time step: 0.002 ps
NPT production run: 200 ns



Sequence of the system to be built

GFP:
VSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLKFICTTGKLPVPWPTLVTTLTYGVQC FSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDDGNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHK LEYNYNSHNVYIMADKQKNGIKVNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKD PNEKRDHMVLLEFVTAAGIT

GBP(N):
DVQLQESGGGSVQAGEALRLSCVGSGYTSINPYMAWFRQAPGKEREGVAAISSGGQYTYYADSVKGRFTI SRDNAKNTMYLQMPSLKPDDSAKYYCAADFRRGGSWNVDPLRYDYQHWGQGTQVTVSS

GBP(C):
DVQLQESGGGSVQAGGSLRLSCAASGFPFSNYCMGWFRQAPGKEREGVATISRLGMFTEYADSVQGRFII SRDNAQNMVFLQMNNLTPEDTAIYYCAAVSTSSSDCRPRLPSQEYTYWGQGTQVTVSSQ

GBP(N) Linker:
SAGGGGGSNAVGQDTQEVIVVP

GBP(C) Linker:
SAGGNAVGQDTQEVIVVP

Helix:
HSLPFKVVVISAILALVVLTIISLIILIMLWQKKPRYE



VMD (Visual Molecular Dynamics)

  VMD is a computer program designed for visualizing the result of molecular dynamics simulations, especially for protein and lipid bilayer system. Also, VMD can animate and analyze the trajectory of a MD simulation then shows molecules in variety drawing methods and materials. So we used VMD to present the suggested binding conformation by a 3D model.





Results & Conclusions




After 200ns simulation, the suggested binding conformation of the complex is shown above. Here in the videos, we showed the N-terminal GBP (showed in yellow) and C-terminal GBP (showed in pink) anchored on the cell membrane separately. At the presence of GFP (showed in green), dimerization of the N-terminal and C-terminal GBP are formed when they are pulled together. Based on the results of the simulation that approve the design of our sensing module, we ran further experiments and confirm the complete design for BioWatcher cells.





Reference:

1. Altintas, Z., and Tothill, I. (2013). Biomarkers and biosensors for the early diagnosis of lung cancer. Sensors Actuators, B Chem. 188, 988–998.