Substrate-based probes

Another important class of substrate-based probes for proteases uses two or more fluorophores, that are self-quenched when in close proximity. " Multiple fluorophores can be linked to graft polymers containing peptide substrate sequences. When these linkers are cleaved by the protease, free fluorescent monomer can be detected. This class of probes has been widely used to study the activity of the cysteine cathepsin family of proteases aeross many diverse disease models. 

Fig. 6.13. Different designs of FRET sensors. (A) Substrates for hydrolytic enzymes. (B) Sensors for bond formation. (C) Sensors based on conformational or structural change. (D) Environmentally sensitive probes. (E) Quenched activity-based probe to monitor small molecule-enzyme interaction. (F) Small molecule-enzyme interaction using a labeled protein.

The greatly increased nucleophilicity of the catalytic serine distinguishes it from all other serine residues and makes it an ideal candidate for modification via activity-based probes [58]. Of the electrophilic probe types to profile serine hydrolases, the fluorophosphonate (FP)-based probes are the most extensively used and were first introduced by Cravatt and coworkers [38, 39]. FPs have been well-known inhibitors of serine hydrolases for over 80 years and were first applied as chemical weapons as potent acetylcholine esterase inhibitors. As FPs do not resemble a peptide or ester substrate, they are nonselective towards a particular serine hydrolase, thus allowing the entire family to be profiled. FPs also show minimal cross-reactivity with other classes of hydrolases such as cysteine-, metallo-, and aspartylhydrolases [59]. Furthermore, FP-based probes react only with the active serine hydrolase, and not the inactive zymogen, allowing these probes to interact only with functional species within the proteome [59]. Extensive use of this probe family has demonstrated their remarkable selectivity for serine hydrolases and resulted in the identification of over 100 distinct serine hydrolases... 

Barglow KT, Cravatt BF (2006) Substrate mimicry in an activity-based probe that targets the nitrilase family of enzymes. Angew Chem Int Ed Engl 45 7408-7411... 

The specificity and affinity of interactions between target molecules bound to the microarray substrate and probe molecules in solution largely determine the quality of microarray assays. The complementary base hybridization is the most efficient and reproducible target-probe interactions used in DNA microarray analysis. 

Measurements of transcript abundance using the Affymetrix GeneChip platform is based upon probe sets consisting of eleven overlapping 25-mer oligonucleotide pairs arrayed on a silicon substrate. Each probe is arranged... 

Inversion at the metal center can be accomplished by any complete metathesis event. To probe whether the catalytically relevant epimerization event was substrate-based, a set of isotopic exchange experiments was performed... 

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