Molecular Biology

ATPases represent a diverse class of enzymes that utilize ATP hydrolysis to support critical biological functions such as driving ion pumps, providing mechanical work, unfolding/folding proteins, and supporting otherwise thermodynamically unfavorable chemical reactions. We have recently shown that the Shigella protein Spa47 is an ATPase that supports protein secretion through its specialized type three secretion apparatus (T3SA), supporting infection of human host cells. Characterizing ATPases, such as Spa47, requires a means to accurately determine enzyme activity (ATP hydrolysis) as a function of time, reaction conditions, and potential cofactors, regulators, inhibitors, etc. Here, we describe a detailed protocol for characterizing the enzyme kinetics of Spa47 using a direct α-³²P ATPase assay.

Protein kinase R (PKR) is one of the key RNA-activated sensors for innate immunity. PKR is activated by pathogenic or aberrant RNAs such as short double-stranded RNAs or those with imperfect secondary structures, as well as a reduction in the amount and number of RNA modifications. Activation of PKR may be an underlying mechanism for the pathogenesis of human diseases. In this protocol, I describe a method for studying levels of RNA-induced PKR activation in vitro.