A genetically encoded photoproximity labeling approach for mapping protein territories.

Studying dynamic biological processes requires approaches compatible with the lifetimes of the biochemical transactions under investigation, which can be very short. We describe a genetically encoded system that allows protein neighborhoods to be mapped using visible light. Our approach involves fusing an engineered flavoprotein to a protein of interest. Brief excitation of the fusion protein leads to the labeling of nearby proteins with cell-permeable probes. Mechanistic studies reveal different labeling pathways are operational depending on the nature of the exogenous probe that is employed. When combined with quantitative proteomics, this photoproximity labeling system generates "snapshots" of protein territories with high temporal and spatial resolution. The intrinsic fluorescence of the fusion domain permits correlated imaging and proteomics analyses, a capability that is exploited in several contexts, including defining the protein clients of the major vault protein. The technology should be broadly useful in the biomedical area.

Comparative study of two Saccharomyces cerevisiae strains with kinetic models at genome-scale.

The parameterization of kinetic models requires measurement of fluxes and/or metabolite levels for a base strain and a few genetic perturbations thereof. Unlike stoichiometric models that are mostly invariant to the specific strain, it remains unclear whether kinetic models constructed for different strains of the same species have similar or significantly different kinetic parameters. This important question underpins the applicability range and prediction limits of kinetic reconstructions. To this end, herein we parameterize two separate large-scale kinetic models using K-FIT with genome-wide coverage corresponding to two distinct strains of Saccharomyces cerevisiae: CEN.PK 113-7D strain (model k-sacce306-CENPK), and growth-deficient BY4741 (isogenic to S288c; model k-sacce306-BY4741). The metabolic network for each model contains 306 reactions, 230 metabolites, and 119 substrate-level regulatory interactions. The two models (for CEN.PK and BY4741) recapitulate, within one standard deviation, 77% and 75% of the fitted dataset fluxes, respectively, determined by 13C metabolic flux analysis for wild-type and eight single-gene knockout mutants of each strain. Strain-specific kinetic parameterization results indicate that key enzymes in the TCA cycle, glycolysis, and arginine and proline metabolism drive the metabolic differences between these two strains of S. cerevisiae. Our results suggest that although kinetic models cannot be readily used across strains as stoichiometric models, they can capture species-specific information through the kinetic parameterization process.

Protein Editing using a Concerted Transposition Reaction.

Protein engineering through the chemical or enzymatic ligation of polypeptide fragments has proven enormously powerful for studying countless biochemical processes in vitro. In general, this strategy necessitates a protein folding step following ligation of the unstructured fragments, a requirement that constrains the types of systems amenable to the approach. Here, we report an in vitro strategy that allows internal regions of target proteins to be replaced in a single operation. Conceptually, our system is analogous to a DNA transposition reaction, but employs orthogonal pairs of split inteins to swap out a designated region of a host protein with an exogenous molecular cassette. We show using isotopic labeling experiments that this 'protein transposition' reaction is concerted when the kinetics for the embedded intein pairs are suitably matched. Critically, this feature allows for efficient manipulation of protein primary structure in the context of a native fold. The utility of this method is illustrated using several protein systems including the multisubunit chromatin remodeling complex, ACF, where we also show protein transposition can occur in situ within the cell nucleus. By carrying out a molecular 'cut and paste' on a protein or protein complex under native folding conditions, our approach dramatically expands the scope of protein semisynthesis.

Cell surface sculpting using logic-gated protein actuators.

Cell differentiation and tissue specialization lead to unique cellular surface landscapes and exacerbated or loss of expression patterns can result in further heterogenicity distinctive of pathological phenotypes1-3. Immunotherapies and emerging protein therapeutics seek to exploit such differences by engaging cell populations selectively based on their surface markers. Since a single surface antigen rarely defines a specific cell type4,5, the development of programmable molecular systems that integrate multiple cell surface features to convert on-target inputs to user-defined outputs is highly desirable. Here, we describe an autonomous decision-making protein device driven by proximity-gated protein trans-splicing that allows local generation of an active protein from two otherwise inactive fragments. We show that this protein actuator platform can perform various Boolean logic operations on cell surfaces, allowing highly selective recruitment of enzymatic and cytotoxic activities to specific cells within mixed populations. Due to its intrinsic modularity and tunability, this technology is expected to be compatible with different types of inputs, targeting modalities and functional outputs, and as such will have broad application in the synthetic biology and biotechnology areas.

Author Correction: H3K14me3 genomic distributions and its regulation by KDM4 family demethylases.

We apologize for an error introduced during format conversion in the paper published online on 18 October 2018. The resolution of the MALDI-TOF result in Fig. 1e underwent an unexpected reduction when transformed from Powerpoint format. The corrected Fig. 1e is provided below on the left.