To determine the most conserved substitutions for free cysteines in the binding proteins, amino acid sequences were inserted into Phyre2, an online server for protein homology which runs an intensive BLAST search, giving a sequence homology output in FASTA format. Sequences for phosphate binding protein, potassium binding protein, glucose/galactose binding protein, alpha-klotho, and the parathyroid hormone receptor (N-termini) were inserted.

Compared to other binding proteins, alpha-klotho had the most free cysteines, requiring the most substitutions. Interestingly, glucose, potassium, and the parathyroid hormone receptor N-termini had no free cysteines, so only one substitution was made – inserting a cysteine to later bind to a coil. Both glucose and potassium binding proteins had their C-termini trimmed by 23 and 8 amino acids, respectively, a recommendation found in literature. To read more about how we mutated, trimmed, and identified residues for cysteine type immobilization refer to our protocol section.

After substitutions took place, mNeonGreen and mCherry were added to the N- and C- terminis of our binding proteins respectively, to allow for fluorescent detection of biomarkers. With phosphate binding protein, a 2 amino acid glycine-threonine linker was inserted between the fluorophores and binding protein, a recommendation from literature. It is worth noting that the parathyroid hormone receptor and alpha-klotho only have a N- termini fluorophore attached. This is because they are involved in competition type assays, with either PTH or FGF23 tagged with the partner fluorophore. More information on this may be found in our contributions or protocol pages.

Fluorescent binding protein constructs with mutations. Cysteine based substitutions were made after intensive homology searches for five biomarker binding proteins. From top to bottom; phosphate, potassium, glucose, PTH, and FGF23 binding proteins. Fluorophore(s) were added to the N- and C- termini’s. DNA basic and composite part sequences may be found on the ‘parts page’ on this wiki or the Registry of Standard Biological Parts.

The one remaining free cysteine on the surface of the binding protein was placed in a position opposite to the active site and away from the fluorophore tails, as to not interfere with either ligand binding or fluorescent activity. Cysteines were not added to areas of intrinsic disorder, as to avoid a binding that may be uncontrolled in spatial space, and further, avoid hindering protein conformational adjustments. Binding proteins with this cysteine were modelled using PyMOL, a molecular viewing software, to ensure that the substitution and cysteine would face away from the protein, towards solvent, and was free of steric clash. Interestingly, all of our cysteines were mutated onto an alpha helix residue facing towards solvent, regardless of the binding protein.

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).

Using Chimera, a protein modeling software, mNeonGreen and mCherry fluorophores were attached the N- and C- terminis, respectively, with the exception of the parathyroid hormone receptor and alpha-klotho. This was to ensure that the cysteine placement did not interfere with the fluorophores in 3D space – as this would either hinder fluorescence, coil binding, or both. A cartoon model preset was generated for reference while a ball and stick preset were used to observe all residues in 3D space. For all proteins, no interference was observed between any potential cysteine placement and the fluorophores.

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.

Potassium 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. Potassium 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.

Glucose/Galactose 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. Glucose/Galactose 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.

Parathyroid receptor hormone 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) and binding protein (blue), are both displayed using PyMOL. A. Parathyroid hormone receptor with a cartoon preset coupled with a fluorophore (mNeonGreen). B. Construct in a stick preset to provide insights regarding the construct volume in 3D space.