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

Modeling

Modeling大標字體大小未與Design、Biosafety一致，未放影片，圖片要拿掉嗎?

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 (cite references), 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.

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-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.

Methodology

　 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).

Molecular Dynamics Simulation

　 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.

　 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.(Fig. The blue part is the GFP chain, the red part is GBP(N) chain,and the gary part is GBP(C) chain.)要放圖片和這句嗎?

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

GBPN: DVQLQESGGGSVQAGEALRLSCVGSGYTSINPYMAWFRQAPGKEREGVAAISSGGQYTYYADSVKGRFTI SRDNAKNTMYLQMPSLKPDDSAKYYCAADFRRGGSWNVDPLRYDYQHWGQGTQVTVSS

GBPC: DVQLQESGGGSVQAGGSLRLSCAASGFPFSNYCMGWFRQAPGKEREGVATISRLGMFTEYADSVQGRFII SRDNAQNMVFLQMNNLTPEDTAIYYCAAVSTSSSDCRPRLPSQEYTYWGQGTQVTVSSQ

GBPN Linker:
SAGGGGGSNAVGQDTQEVIVVP

GPBC Linker:
SAGGNAVGQDTQEVIVVP

Helix:
HSLPFKVVVISAILALVVLTIISLIILIMLWQKKPRYE

VMD(Visual Molecular Dynamics

　 To present the suggested binding conformation clearly, we used VMD to show a 3D model.

Results&Conclusions

　 After 200ns simulation, the suggested binding conformation of the complex is shown below.







　 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 further experiments and confirm the complete design or BioWatcher cells.