Deciphering Design Principles of Förster ResonanceEnergy Transfer-Based Protease Substrates: Thermolysin-Like Proteasefrom Geobacillus stearothermophilus as a Test Case

摘要

Protease activity is frequently assayed using short peptides that are equipped with a Förster resonance energy transfer (FRET) reporter system. Many frequently used donor–acceptor pairs are excited in the ultraviolet range and suffer from low extinction coefficients and quantum yields, limiting their usefulness in applications where a high sensitivity is required. A large number of alternative chromophores are available that are excited in the visible range, for example, based on xanthene or cyanine core structures. These alternatives are not only larger in size but also more hydrophobic. Here, we show that the hydrophobicity of these chromophores not only affects the solubility of the resulting FRET-labeled peptides but also their kinetic parameters in a model enzymatic reaction. In detail, we have compared two series of 4–8 amino acid long peptides, designed to serve as substrates for the thermolysin-like protease (TLP-ste) from Geobacillus stearothermophilus. These peptides were equipped with a carboxyfluorescein donor and either Cy5 or its sulfonated derivative Alexa Fluor 647 as the acceptor. We show that the turnoverrate kcat is largely unaffected by thechoice of the acceptor fluorophore, whereas the KM value is significantly lower for the Cy5- than for theAlexa Fluor 647-labeled substrates. TLP-ste is a rather nonspecificprotease with a large number of hydrophobic amino acids surroundingthe catalytic site, so that the fluorophore itself may form additionalinteractions with the enzyme. This hypothesis is supported by theresult that the difference between Cy5- and Alexa Fluor 647-labeledsubstrates becomes less pronounced with increasing peptide length,that is, when the fluorophore is positioned at a larger distance fromthe catalytic site. These results suggest that fluorophores may becomean integral part of FRET-labeled peptide substrates and that KM and kcat valuesare generally only valid for a specific combination of the peptidesequence and FRET pair.