A batch screening technique for the calculation of chromatographic separability.

This paper employs a high-throughput parallel batch (microtiter plate) adsorption screen with sequential salt step increases to rapidly generate protein elution profiles for multiple resins at different pHs using a protein library. The chromatographic set used in this work includes single mode, multimodal anion-exchange (MMA), and multimodal cation-exchange (MMC) resins. The protein library consists of proteins with isoelectric points ranging from 5.

Identification of the nucleotide-free state as a therapeutic vulnerability for inhibition of selected oncogenic RAS mutants.

RAS guanosine triphosphatases (GTPases) are mutated in nearly 20% of human tumors, making them an attractive therapeutic target. Following our discovery that nucleotide-free RAS (apo RAS) regulates cell signaling, we selectively target this state as an approach to inhibit RAS function. Here, we describe the R15 monobody that exclusively binds the apo state of all three RAS isoforms in vitro, regardless of the mutation status, and captures RAS in the apo state in cells.

Targeting the KRAS α4-α5 allosteric interface inhibits pancreatic cancer tumorigenesis.

is the most frequently mutated oncogene in human cancer with nearly ~20% of cancer patients possessing mutations in one of three genes ( or ). However, is mutated in nearly 90% of pancreatic ductal carcinomas (PDAC). Although pharmacological inhibition of RAS has been challenging, KRAS(G12C)-specific inhibitors have recently entered the clinic.

Conformational interconversion of MLKL and disengagement from RIPK3 precede cell death by necroptosis.

Phosphorylation of the MLKL pseudokinase by the RIPK3 kinase leads to MLKL oligomerization, translocation to, and permeabilization of, the plasma membrane to induce necroptotic cell death. The precise choreography of MLKL activation remains incompletely understood. Here, we report Monobodies, synthetic binding proteins, that bind the pseudokinase domain of MLKL within human cells and their crystal structures in complex with the human MLKL pseudokinase domain.

The structural basis of promiscuity in small multidrug resistance transporters.

By providing broad resistance to environmental biocides, transporters from the small multidrug resistance (SMR) family drive the spread of multidrug resistance cassettes among bacterial populations. A fundamental understanding of substrate selectivity by SMR transporters is needed to identify the types of selective pressures that contribute to this process. Using solid-supported membrane electrophysiology, we find that promiscuous transport of hydrophobic substituted cations is a general feature of SMR transporters.

Identification of MLKL membrane translocation as a checkpoint in necroptotic cell death using Monobodies.

The necroptosis cell death pathway has been implicated in host defense and in the pathology of inflammatory diseases. While phosphorylation of the necroptotic effector pseudokinase Mixed Lineage Kinase Domain-Like (MLKL) by the upstream protein kinase RIPK3 is a hallmark of pathway activation, the precise checkpoints in necroptosis signaling are still unclear. Here we have developed monobodies, synthetic binding proteins, that bind the N-terminal four-helix bundle (4HB) "killer" domain and neighboring first brace helix of human MLKL with nanomolar affinity.