Deciphering Design Principles of Förster Resonance Energy Transfer-Based Protease Substrates: Thermolysin-Like Protease from Geobacillus stearothermophilus as a Test Case

Sophie Ripp,Alan E Rowan,Petri Turunen,Ethan D Minot,Kerstin G Blank

doi:10.1021/acsomega.7b02084

Abstract

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 turnover rate kcat is largely unaffected by the choice of the acceptor fluorophore, whereas the KM value is significantly lower for the Cy5- than for the Alexa Fluor 647-labeled substrates. TLP-ste is a rather nonspecific protease with a large number of hydrophobic amino acids surrounding the catalytic site, so that the fluorophore itself may form additional interactions with the enzyme. This hypothesis is supported by the result that the difference between Cy5- and Alexa Fluor 647-labeled substrates becomes less pronounced with increasing peptide length, that is, when the fluorophore is positioned at a larger distance from the catalytic site. These results suggest that fluorophores may become an integral part of FRET-labeled peptide substrates and that KM and kcat values are generally only valid for a specific combination of the peptide sequence and FRET pair.

Highlights

Fluorescent reporter systems for the detection of protease activity consist of a spectroscopic probe with cleavable bonds
The fluorescence properties of the probe are altered, which allows for following the enzymatic reaction over time.[1−8] Three fundamentally different designs are usually utilized for the detection of protease activity: The first design consists of a fluorophore core carrying one or multiple peptide chain(s) coupled to the fluorophore via a peptide bond.[3,7−12]
Our results suggest that both the peptide length and the nature of the acceptor fluorophore determine the kinetic constants of the enzymatic reaction

Summary

Introduction

Fluorescent reporter systems for the detection of protease activity consist of a spectroscopic probe with cleavable bonds. These substrates are highly artificial, as the fluorophore replaces the C-terminal part of the substrate (P1′−P4′ subsites, according to the nomenclature of Schechter and Berger[13]) These substrates often carry two enzyme cleavable bonds,[7,8] and the reaction proceeds via an intermediate with different photophysical properties, so that the reaction velocity cannot be determined.[11] The second design makes use of selfquenching between a large number of fluorophores coupled to a model protein.[5,6] in this case, the substrate is hardly fluorescent, and the fluorescence signal increases once the peptide fragments are released. In contrast to the first design, the peptide sequence is chosen such that it Received: December 29, 2017 Accepted: March 19, 2018 Published: April 12, 2018

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