Volume 10 Issue 3, March 2014

Ryan Mehl is on a mission to make unnatural amino acids part of the natural order at the Unnatural Protein Facility.

The bacterial halogenase SyrB2 catalyzes selective installation of halogens in place of unactivated aliphatic C-H bonds. By substituting halide reagents with the nitrogenous anions N₃⁻ and NO₂⁻, SyrB2 can perform C-N bond formation reactions not previously observed in nature.

TRPM3 can permeate ions through two distinct pores—the central pore and a likely alternative 'omega pore'. Entry of ions through the alternative pore of TRPM3 contributes to pain generation, making it an attractive target for the design of new analgesics.

System-wide approaches are gaining popularity over traditional studies of individual molecular components. A new NMR approach unravels the interaction hierarchy of these different components by placing them in direct competition for the same target.

A stable replication system, based on a DNA polymerase that replicates cytoplasmic plasmids with unique terminal proteins, decouples plasmid gene replication from host genome replication, allowing for continuous targeted evolution in vivo.

Translational reprogramming, which enables site-specific incorporation of non-natural amino acids into proteins, offers practical tools for studying protein function but also provides insights into the genetic code. Bacteriophages engineered with a 21-amino-acid genetic code make use of the additional noncanonical amino acid during in vitro evolution.

Allosteric small-molecule inhibitors of the p53-induced oncogenic phosphatase Wip1 bind at a ‘flap’ subdomain near the catalytic site. Inhibitor GSK2830371 inhibits the phosphatase and oncogenic activities in cell lines and a xenograft tumor model.

Besides the canonical pore that allows passage of cations into the cell, the TRPM3 channel has an alternative ‘omega’ permeation pathway formed by the S1–S4 region of the protein. This peripheral pore is inwardly rectifying, whereas the canonical central axis pore is outwardly rectifying.

Optogenetic systems permit the temporal and spatial control of gene expression using light. A variant of the LOV domain–containing EL222 protein displays responsive blue light–gated transcriptional control of genes in zebrafish and in mammalian cell lines.

Synthetic biology requires orthogonal inputs and outputs to avoid undesired crosstalk between genetic constructs. Transcription activator–like effectors (TALEs), which bind diverse DNA sequences and can thus be orthogonal, are now employed to construct NOR gates and logic circuits in mammalian cells.

Halogenases differ from hydroxylases by coordination of a chloride ion at the reactive iron center, which is taken up by an activated substrate. Biochemical and spectroscopic evidence now show other anions can be used, resulting in the first enzymatic incorporation of nitrogen onto unactivated aliphatic carbons.

Phage-assisted continuous evolution (PACE) minimizes researcher intervention while maximizing rounds of protein evolution. New strategies now eliminate the need for intermediate substrate analogs and promote altered selectivity instead of promiscuity, exemplified by a 10,000-fold switch in polymerase specificity while retaining wild-type activity.

An NMR analysis of RAS-binding domains (effectors) placed under direct competition allows a systems-level view of effector binding and describes how oscillating concentrations can lead to effector switching. Deconvolution of effector binding to oncogenic RAS^G12V revealed a reordering of the effector hierarchy.

A tethered ligand approach reveals that calcium-binding proteins (CaBPs) act as allosteric modulators of calcium channel calmodulin regulation, shedding light on how trace CaBPs can prevail over an abundance of CaM.