Initial Kinetic Characterization of Sterile Alpha and Toll/Interleukin Receptor Motif-Containing Protein 1.

Sterile alpha and toll/interleukin receptor (TIR) motif-containing protein 1 (SARM1) plays a pivotal role in triggering the neurodegenerative processes that underlie peripheral neuropathies, traumatic brain injury, and neurodegenerative diseases. Importantly, SARM1 knockdown or knockout prevents degeneration, thereby demonstrating that SARM1 is a promising therapeutic target. Recently, SARM1 was shown to promote neurodegeneration via its ability to hydrolyze NAD+, forming nicotinamide and ADP ribose (ADPR). Herein, we describe the initial kinetic characterization of full-length SARM1, as well as the truncated constructs corresponding to the SAM1-2TIR and TIR domains, highlighting the distinct challenges that have complicated efforts to characterize this enzyme. Moreover, we show that bacterially expressed full-length SARM1 (kcat/KM = 6000 ± 2000 M-1 s-1) is at least as active as the TIR domain alone (kcat/KM = 1500 ± 300 M-1 s-1). Finally, we show that the SARM1 hydrolyzes NAD+ via an ordered uni-bi reaction in which nicotinamide is released prior to ADPR.

Rational conversion of affinity reagents into label-free sensors for Peptide motifs by designed allostery.

Optical biosensors for short peptide motifs, an important class of biomarkers, have been developed based on "affinity clamps", a new class of recombinant affinity reagents. Affinity clamps are engineered by linking a peptide-binding domain and an antibody mimic domain based on the fibronectin type III scaffold, followed by optimization of the interface between the two. This two-domain architecture allows for the design of allosteric coupling of peptide binding to fluorescence energy transfer between two fluorescent proteins attached to the affinity clamp. Coupled with high affinity and specificity of the underlying affinity clamps and rationally designed mutants with different sensitivity, peptide concentrations in crude cell lysate were determined with a low nanomolar detection limit and over 3 orders of magnitude. Because diverse affinity clamps can be engineered, our strategy provides a general platform to generate a repertoire of genetically encoded, label-free sensors for peptide motifs.