Our team spent a fair bit of time searching the RCSB Protein Data Bank and literature, looking for the proteins to bind our biomarkers with a high degree of specificity. All three phosphate, potassium, and glucose binding proteins undergo a conformational change as they bind their ligand, alongside PTH and the PTH receptor, have been proven in literature to work with FRET.
Phosphate binding protein fluorescent construct. Protein structures were obtained from the RCSB Protein Data Bank and a construct was made using Chimera software. Torsion angles between fluorescent and binding proteins are adjusted for display purposes.. mNeonGreen (green), binding protein (blue), and mCherry (red) are all displayed using PyMOL. A. Phosphate binding protein with a cartoon preset coupled with two fluorophores (mNeonGreen and mCherry). B. Construct in a stick preset to provide insights regarding the construct volume in 3D space.
While PTH and FGF23 receptors do not undergo a conformational change, we developed a FRET based competition assay to measure concentrations of these biomarkers. For more information see our contributions page. Using this novel method, a peptide hormone from the body can replace a fluorophore tagged peptide hormone in the biosensor – a drop in fluorescence intensity which can make hormone concentration readily quantifiable.
Parathyroid hormone receptor competitive fluorescent assay. Protein structures were obtained from the RCSB Protein Data Bank. All protein residues are shown in a cartoon preset with the mNeonGreen, mCherry, parathyroid hormone receptor, and parathyroid hormone (1-34) as green, red, blue, and grey, respectively, using Chimera software. A. Parathyroid receptor hormone with mNeonGreen and parathyroid hormone with mCherry interaction. Fluorescent is expected to occur. B. Parathyroid hormone from the interstitial fluid enters the biosensor and competes with the parathyroid hormone bound to mCherry. Interaction with parathyroid hormone receptor with mNeonGreen. Quantifiable drop in fluorescent intensity expected.
The most important rule in our method of protein immobilization is that the constructs protein binding component must have one cysteine residue. The reasoning behind this is that our coil must form a disulfide bridge with a cysteine residue in an optimal orientation that allows ligand binding and fluorescence to occur. If there is more than one cysteine, binding cannot be controlled and association with multiple coils may occur.
Cysteine immobilization modifications of binding proteins. Protein structures were obtained from the RCSB Protein Data Bank. All protein residues are shown in a blue cartoon preset with cysteine residues in red cartoon, respectively, using PyMOL. A. Phosphate binding protein with a Cys 256 modification (red). B. Potassium binding protein with a Cys 26 modification (red). C. Parathyroid hormone receptor, with a Cys 48 residue (red). D. Glucose/Galactose binding protein with a Cys 190 modification (red). E. Alpha-klotho (FGF23 receptor) with a Cys 664 modification (red).
And another important rule – specificity. If the binding protein binds multiple ligands in its active site, it may make quantifying a biomarker concentration uncertain. Before using any protein, we ensured that it was tested in literature to work with FRET and that it only bound its correct substrate. With the exception of FGF23, our proteins in our biosensor have been carefully vetted as specificity is one of the most important considerations.
Alpha-Klotho (FGF23 receptor) protein fluorescent construct electrostatic potential map. Protein structures were obtained from the RCSB Protein Data Bank and a construct was made using Chimera software. Torsion angles between fluorescent and binding proteins are adjusted for display purposes. mNeonGreen and binding protein are oriented left to right in the ‘FRONT’ figure and were generated using the APBS Electrostatics function in PyMOL. 3D volume in space can also be visualized. A high and low electrostatic potential is indicated by blue and red, respectively. Note: A high electrostatic potential indicates an absence of electrons and is positive.
To learn more about the design of our binding proteins, including how we immobilize proteins, visit our protocol page.