Structural Biochemistry/Detection of Phosphorylation

Peptide Biosensor
Peptide biosensors are used to determine phosphorylation activities in cells. A biosensor is a device that provides information about the composition, structure and function of biological analytes such as isolated enzymes, immonosystems, tissues, and organelles by converting a biological response into an electrical, thermal, or optical signals. The biosensors that are mainly used for the analysis of phosphorylation events incorporate the use of a synthetic fluorophore, the portion of a molecule that illuminates the color of the molecule. When the fluorophore interacts with a phosphorylated peptide or protein domain, the fluorescence properties of the complex changes. Fluorscence are dyes that emit a certain color when a molecule is exposed to light. In general, the emitted light is usually longer than that of the incident light. The complex causes an enhancement in fluorescence and this property is used to determine the phosphorylation event of an analyte.

Environmentally Sensitive Biosensor
An environmentally sensitive biosensor detects phosphorylation activities that occur between a solute and a solvent molecule that forms relatively weak covalent bonds. In general, a domain that has a high affinity for binding to a phosphospecific amino acid attaches to a phosphorylated peptide or protein. This complex changes the polarity of the solvent in the fluorophore which increases the fluorescence of the molecule and the complex illuminates a noticeable color.

Deep-Quench Biosensor
A deep-quench biosensor utilizes a quencher that interacts with the fluorophore in the peptide. When this complex is phosphorylated, the biosensor employs a domain that has a high affinity for binding to a phosphospecific amino acid to separate the quencher from the fluorophore. This separation causes an increase in the fluorescence in the molecule.

Self-Reporting Sensors
A self-reporting sensors is a type of sensor that does not require the use of a domain that has a high affinity for binding to a phosphospecific amino acid in order to detect the presence of phosphorylated amino acid. This type of sensor is only applicable for amino acids that are aromatic, usually tyrosine. The pi bonds in the molecule enable the amino acid to quench the fluorophore in the peptide biosensor. When the molecular is phosphorylated, the aromatic amino acid cannot quench the flouorophore again.

Metal Chelation-Enhanced Fluorescence Biosensors
Metal chelation-enhanced fluorescence detects the presence of a phosphorylation event via Sox, a gene that encodes for a transcription factor that binds to the DNA. In addition being a sequence known the HMG box, Sox is also a fluorophore which can be intensified by chelation. Due to this unique property, Sox is used to determined phosphorylation activities in a system. In addition, metal chelation-enhanced fluorescence do not require the use of an acid-binding domain that has a high affinity for a phosphoamino acid. In the presence of a serine or threonine residue, Sox is capable of recruiting a phosphate group to gather, which elicits phosphorylation to occur. Once the serine or threonine residue has been phosphorylated via Sox, the peptide acquires a higher ability to bind to Mg2+ peptides. The binding of the Mg2+ to the phosphorylated serine or threonine residue generates a fluorescent signal. In addition, several probes such as PKC, Cdk2, and PKA have been created to increase the intensity of the fluorescent signal.