The ability to routinely engineer protease specificity can allow us to better understand and modulate their biology for expanded therapeutic and industrial applications. Here we report a new approach based on a caged green fluorescent protein (CA-GFP) reporter that allows for flow-cytometry-based selection in bacteria or other cell types enabling selection of intracellular protease specificity, regardless of the compositional complexity of the protease. Here we apply this approach to introduce the specificity of caspase-6 into caspase-7, an intracellular cysteine protease important in cellular remodeling and cell death. We found that substitution of substrate-contacting residues from caspase-6 into caspase-7 was ineffective, yielding an inactive enzyme, whereas saturation mutagenesis at these positions and selection by directed evolution produced active caspases. The process produced a number of non-obvious mutations that enabled conversion of the caspase-7 specificity to match caspase-6. The structures of the evolved-specificity caspase-7 (esCasp-7) revealed alternate binding modes for the substrate, including reorganization of an active site loop. Profiling the entire human proteome of esCasp-7 by N-terminomics demonstrated that the global specificity toward natural protein substrates is remarkably similar to that of caspase-6. Because the esCasp-7 maintained the core of caspase-7, we were able to identify a caspase-6 substrate, lamin C, that we predict relies on an exosite for substrate recognition. These reprogrammed proteases may be the first tool built with the express intent of distinguishing exosite dependent or independent substrates. This approach to specificity reprogramming should also be generalizable across a wide range of proteases.

Proteases are one of the most important and historically utilized classes of drug targets. To effectively interrogate this class of proteins, which encodes nearly 2% of the human proteome, it is necessary to develop effective and cost-efficient methods that report on their activity both in vitro and in vivo. We have developed a robust reporter of caspase proteolytic activity, called caspase-activatable green fluorescent protein (CA-GFP). The caspases play central roles in homeostatic regulation, as they execute programmed cell death, and in drug design, as caspases are involved in diseases ranging from cancer to neurodegeneration. CA-GFP is a genetically encoded dark-to-bright fluorescent reporter of caspase activity in in vitro, cell-based, and animal systems. Based on the CA-GFP platform, we developed reporters that can discriminate the activities of caspase-6 and -7, two highly related proteases. A second series of reporters, activated by human rhinovirus 3C protease, demonstrated that we could alter the specificity of the reporter by reengineering the protease recognition sequence. Finally, we took advantage of the spectrum of known fluorescent proteins to generate green, yellow, cyan, and red reporters, paving the way for multiplex protease monitoring.

Water vaporpressure for cornstarch andpopcorn grits at a temperature o f between 100 and 180°C was determined by measuring vapor pressures generated at various moisture contents in a heated, closed system. At a higher temperature, fugacity of water increased with increasing temperature probably because of melting or partial melting of starch. The apparent heat of sorption for cornstarch changedsignificantly with moisture content and temperature. Noncondensible gases were produced by heating popcorn grits to temperatures above 150°C. It is not accurate to estimatevapor pressure based on the heat of sorption or extrapolation of currently used empirical isotherm equations to a temperature above 100" C. Vapor pressure inside popcorn at the instant of popping are roughly 758 to 827 kPa (110 to 120psia).

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Caspases are among the most specific of proteases, making them ideal targets for engineering new specificity and developing new protease‐based biotherapeutics. However, conventional high‐throughput methods for protein evolution are not amenable to caspases as they are multi‐chain, and intracellular. Increasing the complexity, active sites of caspases are highly flexible to allow for accommodation of various substrates, making a rational design approach difficult To overcome these challenges, we developed a method for evolving function in cytosolic proteases, based on our novel genetically‐encoded caspase activatable‐GFP reporter (CA‐GFP), which is activated from a dark to a fluorescent state by proteolysis.Our first application of this technology, we engineered caspase‐7, which cleaves the amino acid sequence DEVD to recognize and cleave the new sequence VEID, recognized by caspase‐6. Saturation mutagenesis at key residues within the active site of the enzyme allowed us to sort for variants with altered specificity by flow cytometry. Variants able to cleave the target sequence were purified, then characterized using traditional Michaelis‐Menten kinetics, protein substrate assays and x‐ray crystallography. We identified several variants that displayed kinetic activity and cleavage patterns similar to the wild type caspase‐6. X‐ray crystal structures of the evolved variants bound to the casp‐6 and ‐7 cognate substrates VEID, and DEVD reveal how the mutations affect the activity of the enzyme, which could not be rationally predicted, emphasizing the strength of this approach. Utilizing this method we are able to reengineer cytosolic proteases to cleave novel targets and pave the way for biotherapeutic applications.This work is supported in part by NIH GM080532.