Identifying peptide substrates that are efficiently cleaved by proteases gives insights into substrate recognition and specificity, guides development of inhibitors, and improves assay sensitivity. Peptide arrays and SAMDI mass spectrometry were used to identify a tetrapeptide substrate exhibiting high activity for the bacterial outer-membrane protease (OmpT). Analysis of protease activity for the preferred residues at the cleavage site (P1, P1') and nearest-neighbor positions (P2, P2') and their positional interdependence revealed FRRV as the optimal peptide with the highest OmpT activity. Substituting FRRV into a fragment of LL37, a natural substrate of OmpT, led to a greater than 400-fold improvement in OmpT catalytic efficiency, with a k /K value of 6.1×10 L mol s . Wild-type and mutant OmpT displayed significant differences in their substrate specificities, demonstrating that even modest mutants may not be suitable substitutes for the native enzyme.

Identifying peptide substrates that are efficiently cleaved by proteases gives insights into substrate recognition and specificity,guides development of inhibitors,and improves assays ensitivity.P eptide arrays and SAMDI mass spectrometry were used to identify at etrapeptide substrate exhibiting high activity for the bacterial outer-membrane protease (OmpT). Analysis of protease activity for the preferred residues at the cleavage site (P1, P1')a nd nearest-neighbor positions (P2, P2')a nd their positional interdependence revealed FRRV as the optimal peptide with the highest OmpT activity.S ubstituting FRRV into af ragment of LL37, an atural substrate of OmpT,l ed to ag reater than 400-fold improvement in OmpT catalytic efficiency,with ak cat /K m value of 6.1 10 6 Lmol À1 s À1 .Wild-type and mutant OmpT displayed significant differences in their substrate specificities,d emonstrating that even modest mutants mayn ot be suitable substitutes for the native enzyme.

Outer membrane protease (OmpT) is a 33.5 kDa aspartyl protease that cleaves at dibasic sites and is thought to function as a defense mechanism for E. coli against cationic antimicrobial peptides secreted by the host immune system. Despite carrying three dibasic sites in its own sequence, there is no report of OmpT autoproteolysis in vivo. However, recombinant OmpT expressed in vitro as inclusion bodies has been reported to undergo autoproteolysis during the refolding step, thus resulting in an inactive protease. In this study, we monitor and compare levels of in vitro autoproteolysis of folded and unfolded OmpT and examine the role of lipopolysaccharide (LPS) in autoproteolysis. SDS-PAGE data indicate that it is only the unfolded OmpT that undergoes autoproteolysis while the folded OmpT remains protected and resistant to autoproteolysis. This selective susceptibility to autoproteolysis is intriguing. Previous studies suggest that LPS, a co-factor necessary for OmpT activity, may play a protective role in preventing autoproteolysis. However, data presented here confirm that LPS plays no such protective role in the case of unfolded OmpT. Furthermore, OmpT mutants designed to prevent LPS from binding to its putative LPS-binding motif still exhibited excellent protease activity, suggesting that the putative LPS-binding motif is of less importance for OmpT’s activity than previously proposed.

E. coli and Salmonella are two of the most common bacterial pathogens involved in foodborne and waterborne related deaths. Hence, it is critical to develop rapid and sensitive detection strategies for near‐outbreak applications. Reported is a simple and specific assay to detect as low as 1 CFU mL−1 of E. coli in water within 6 hours by targeting the bacteria's surface protease activity. The assay relies on polythiophene acetic acid (PTAA) as an optical reporter and a short unlabeled peptide (LL37FRRV) previously optimized as a substrate for OmpT, an outer‐membrane protease on E. coli. LL37FRRV interacts with PTAA to enhance its fluorescence while also inducing the formation of a helical PTAA‐LL37FRRV construct, as confirmed by circular dichroism. However, in the presence of E. coli LL37FRRV is cleaved and can no longer affect the conformations and optical properties of PTAA. This ability to distinguish between an intact and cleaved peptide was investigated in detail using LL37FRRV sequence variants.

Poly(3-alkylthiophene) (PT)-based conjugated polyelectrolytes (CPEs) constitute an important class of responsive polymers with excellent optical properties. The electrostatic interactions between PTs and target analytes trigger complexation and concomitant conformational changes of the PT backbones that produce distinct optical responses. These conformation-induced optical responses of the PTs enable them to be utilized as reporters for detection of various analytes by employing simple UV−vis spectrophotometry or the naked eye. Numerous PTs with unique pendant groups have been synthesized to tailor their interactions with analytes such as nucleotides, ions, surfactants, proteins, and bacterial and viral pathogens. In this perspective, we discuss PTtarget analyte complexation for bioanalytical applications and highlight recent advancements in point-of-care and field deployable assays. Subsequently, we highlight a few areas of critical importance for future applications of PTs as reporters, including (i) design and synthesis of specific PTs to advance the understanding of the mechanisms of interaction with target analytes, (ii) using arrays of PTs and linear discriminant analysis for selective and specific detection of target analytes, (iii) translation of conventional homogeneous solution-based assays into heterogeneous membrane-based assay formats, and finally (iv) the potential of using PT as an alternative to conjugated polymer nanoparticles and dots in bioimaging.