De novo design of tyrosine and serine kinase-driven protein switches.

Kinases play central roles in signaling cascades, relaying information from the outside to the inside of mammalian cells. De novo designed protein switches capable of interfacing with tyrosine kinase signaling pathways would open new avenues for controlling cellular behavior, but, so far, no such systems have been described. Here we describe the de novo design of two classes of protein switch that link phosphorylation by tyrosine and serine kinases to protein-protein association. In the first class, protein-protein association is required for phosphorylation by the kinase, while in the second class, kinase activity drives protein-protein association. We design systems that couple protein binding to kinase activity on the immunoreceptor tyrosine-based activation motif central to T-cell signaling, and kinase activity to reconstitution of green fluorescent protein fluorescence from fragments and the inhibition of the protease calpain. The designed switches are reversible and function in vitro and in cells with up to 40-fold activation of switching by phosphorylation.

Self-assembly-based posttranslational protein oscillators.

Recent advances in synthetic posttranslational protein circuits are substantially impacting the landscape of cellular engineering and offer several advantages compared to traditional gene circuits. However, engineering dynamic phenomena such as oscillations in protein-level circuits remains an outstanding challenge. Few examples of biological posttranslational oscillators are known, necessitating theoretical progress to determine realizable oscillators. We construct mathematical models for two posttranslational oscillators, using few components that interact only through reversible binding and phosphorylation/dephosphorylation reactions. Our designed oscillators rely on the self-assembly of two protein species into multimeric functional enzymes that respectively inhibit and enhance this self-assembly. We limit our analysis to within experimental constraints, finding (i) significant portions of the restricted parameter space yielding oscillations and (ii) that oscillation periods can be tuned by several orders of magnitude using recent advances in computational protein design. Our work paves the way for the rational design and realization of protein-based dynamic systems.

Author Correction: A cell-free platform for the prenylation of natural products and application to cannabinoid production.

In the original version of this Article, the genotype of the M30 mutant presented in Fig. 3b was given incorrectly as Y288V/A232S, and the M31 mutant was given incorrectly as M1/A232S. The correct genotype of the M30 mutant is Y288A/A232S and for M31 it is Y288V/A232S. In addition, to keep consistency in genotype formatting, the genotype of the M27 mutant should be Y288V/G286S. The errors have been corrected in both the PDF and HTML versions of the Article.

A cell-free platform for the prenylation of natural products and application to cannabinoid production.

Prenylation of natural compounds adds structural diversity, alters biological activity, and enhances therapeutic potential. Because prenylated compounds often have a low natural abundance, alternative production methods are needed. Metabolic engineering enables natural product biosynthesis from inexpensive biomass, but is limited by the complexity of secondary metabolite pathways, intermediate and product toxicities, and substrate accessibility. Alternatively, enzyme catalyzed prenyl transfer provides excellent regio- and stereo-specificity, but requires expensive isoprenyl pyrophosphate substrates. Here we develop a flexible cell-free enzymatic prenylating system that generates isoprenyl pyrophosphate substrates from glucose to prenylate an array of natural products. The system provides an efficient route to cannabinoid precursors cannabigerolic acid (CBGA) and cannabigerovarinic acid (CBGVA) at >1 g/L, and a single enzymatic step converts the precursors into cannabidiolic acid (CBDA) and cannabidivarinic acid (CBDVA). Cell-free methods may provide a powerful alternative to metabolic engineering for chemicals that are hard to produce in living organisms.

Complete topology inversion can be part of normal membrane protein biogenesis.

The topology of helical membrane proteins is generally defined during insertion of the transmembrane helices, yet it is now clear that it is possible for topology to change under unusual circumstances. It remains unclear, however, if topology reorientation is part of normal biogenesis. For dual topology dimer proteins such as the multidrug transporter EmrE, there may be evolutionary pressure to allow topology flipping so that the populations of both orientations can be equalized. We previously demonstrated that when EmrE is forced to insert in a distorted topology, topology flipping of the first transmembrane helix can occur during translation. Here, we show that topological malleability also extends to the C-terminal helix and that even complete topology inversion of the entire EmrE protein can occur after the full protein is translated and inserted. Thus, topology rearrangements are possible during normal biogenesis. Wholesale topology flipping is remarkable given the physical constraints of the membrane and expands the range of possible membrane protein folding pathways, both productive and detrimental.

Dual-topology insertion of a dual-topology membrane protein.

Some membrane transporters are dual-topology dimers in which the subunits have inverted transmembrane topology. How a cell manages to generate equal populations of two opposite topologies from the same polypeptide chain remains unclear. For the dual-topology transporter EmrE, the evidence to date remains consistent with two extreme models. A post-translational model posits that topology remains malleable after synthesis and becomes fixed once the dimer forms. A second, co-translational model, posits that the protein inserts in both topologies in equal proportions. Here we show that while there is at least some limited topological malleability, the co-translational model likely dominates under normal circumstances.

Bacteriorhodopsin folds through a poorly organized transition state.

The folding mechanisms of helical membrane proteins remain largely uncharted. Here we characterize the kinetics of bacteriorhodopsin folding and employ φ-value analysis to explore the folding transition state. First, we developed and confirmed a kinetic model that allowed us to assess the rate of folding from SDS-denatured bacteriorhodopsin (bRU) and provides accurate thermodynamic information even under influence of retinal hydrolysis. Next, we obtained reliable φ-values for 16 mutants of bacteriorhodopsin with good coverage across the protein. Every φ-value was less than 0.4, indicating the transition state is not uniquely structured. We suggest that the transition state is a loosely organized ensemble of conformations.

Development of an amine dehydrogenase for synthesis of chiral amines.

A leucine dehydrogenase has been successfully altered through several rounds of protein engineering to an enantioselective amine dehydrogenase. Instead of the wild-type α-keto acid, the new amine dehydrogenase now accepts the analogous ketone, methyl isobutyl ketone (MIBK), which corresponds to exchange of the carboxy group by a methyl group to produce chiral (R)-1,3-dimethylbutylamine.