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Outline
• Introduction : computational method used
• Paper discussion
• Biotechnological applications
Computational design of receptor
and sensor proteins with novel
functions
Nature vol..423
May 8 2003, pp.:185-190
• Designed a computational method:
– identify amino acid sequences predicted to form
a complimentary surface between the protein
and a target ligand replacing the wild type
ligand
Prot ein
coordinat es
1
Poly-alanine
PCS
2
Docking
grid
Ligand
coordinat es
3
Rot at ional
ligand
ensemble
Force field
4
Docked
ligand
ensemble
5
Ranked ligand
ensemble
Side-chain
6
rot amers
Evolved
PCS
ensemble
7
Ranked PCS
ensemble
8
Experiment s
b
• To test their method they chose E.coli.
• Periplasmic binding protein (PBP):
–
–
–
–
–
glucose binding protein (GBP)
ribose binding protein (RBP)
arabinose binding protein (ABP)
glutamine binding protein (QBP)
histidine binding protein (HBP)
• Target ligands bear little resemblance to
wild type and chemically different from
each other.
• To explore critical parameters of molecular
recognition:
– molecular shape, chirality, functional groups,
internal flexibility,charge, and water solubility.
• How were they able to know if there is
binding of the ligand ?
• Introducing fluorophores at residues that
exhibit changes in fluorescence emission
• due to changes in conformation (open vs
close)
Test it in cells
• RBP and GBP involved in signal
transduction pathways at the gene levels
• presence of ligand to the above mentioned
receptors will cause the upregulation of
b-galactosidase gene expression.
• The RBP and GBP were replaced with the
designed TNT and L-lactate receptors and
tested using this expression mechanism .
Biotechnological applications
• Developing specific cell based detectors for
chemical threats and pollutants:
– TNT is carcinogen and explosive, having
bacteria with TNT.R3 that respond with the
expression of a fluorescent gene
– Products such as drugs needs to be highly
purified and with receptors being able to
differentiate between chiral (L-lactate)
compounds pharmaceutical companies will be
able to achieve this goal