Droplet-based screening of phosphate transfer catalysis reveals how epistasis shapes MAP kinase interactions with substrates.

The combination of ultrahigh-throughput screening and sequencing informs on function and intragenic epistasis within combinatorial protein mutant libraries. Establishing a droplet-based, in vitro compartmentalised approach for robust expression and screening of protein kinase cascades (>10 variants/day) allowed us to dissect the intrinsic molecular features of the MKK-ERK signalling pathway, without interference from endogenous cellular components. In a six-residue combinatorial library of the MKK1 docking domain, we identified 29,563 sequence permutations that allow MKK1 to efficiently phosphorylate and activate its downstream target kinase ERK2.

Improved RAD51 binders through motif shuffling based on the modularity of BRC repeats.

Exchanges of protein sequence modules support leaps in function unavailable through point mutations during evolution. Here we study the role of the two RAD51-interacting modules within the eight binding BRC repeats of BRCA2. We created 64 chimeric repeats by shuffling these modules and measured their binding to RAD51.

Error-Free Synthetic DNA by Molecular Dictation.

Synthetic DNA is the linchpin of the rapidly accelerating biotechnological era and is perhaps the most promising candidate for long-term digital data storage. Despite huge advances, manufacturing error-free DNA at low cost and high throughput remains challenging. Borrowing from well-established sequencing-by-synthesis technologies, we describe a new solution for DNA error correction.

"NAD-display": Ultrahigh-Throughput in Vitro Screening of NAD(H) Dehydrogenases Using Bead Display and Flow Cytometry.

NAD(H)-utiliing enzymes have been the subject of directed evolution campaigns to improve their function. To enable access to a larger swath of sequence space, we demonstrate the utility of a cell-free, ultrahigh-throughput directed evolution platform for dehydrogenases. Microbeads (1.

Multiplexed Affinity Characterization of Protein Binders Directly from a Crude Cell Lysate by Covalent Capture on Suspension Bead Arrays.

The precise determination of affinity and specificity is a crucial step in the development of new protein reagents for therapy and diagnostics. Paradoxically, the selection of protein binders, e.g.

Split & mix assembly of DNA libraries for ultrahigh throughput on-bead screening of functional proteins.

Site-saturation libraries reduce protein screening effort in directed evolution campaigns by focusing on a limited number of rationally chosen residues. However, uneven library synthesis efficiency leads to amino acid bias, remedied at high cost by expensive custom synthesis of oligonucleotides, or through use of proprietary library synthesis platforms. To address these shortcomings, we have devised a method where DNA libraries are constructed on the surface of microbeads by ligating dsDNA fragments onto growing, surface-immobilised DNA, in iterative split-and-mix cycles.

Quantifying stickiness: thermodynamic characterization of intramolecular domain interactions to guide the design of förster resonance energy transfer sensors.

The introduction of weak, hydrophobic interactions between fluorescent protein domains (FPs) can substantially increase the dynamic range (DR) of Förster resonance energy transfer (FRET)-based sensor systems. Here we report a comprehensive thermodynamic characterization of the stability of a range of self-associating FRET pairs. A new method is introduced that allows direct quantification of the stability of weak FP interactions by monitoring intramolecular complex formation as a function of urea concentration.

Engineering genetically encoded FRET sensors.

Förster Resonance Energy Transfer (FRET) between two fluorescent proteins can be exploited to create fully genetically encoded and thus subcellularly targetable sensors. FRET sensors report changes in energy transfer between a donor and an acceptor fluorescent protein that occur when an attached sensor domain undergoes a change in conformation in response to ligand binding. The design of sensitive FRET sensors remains challenging as there are few generally applicable design rules and each sensor must be optimized anew.

MagFRET: the first genetically encoded fluorescent Mg2+ sensor.

Magnesium has important structural, catalytic and signaling roles in cells, yet few tools exist to image this metal ion in real time and at subcellular resolution. Here we report the first genetically encoded sensor for Mg(2+), MagFRET-1. This sensor is based on the high-affinity Mg(2+) binding domain of human centrin 3 (HsCen3), which undergoes a transition from a molten-globular apo form to a compactly-folded Mg(2+)-bound state.

Rational design of FRET sensor proteins based on mutually exclusive domain interactions.

Proteins that switch between distinct conformational states are ideal to monitor and control molecular processes within the complexity of biological systems. Inspired by the modular architecture of natural signalling proteins, our group explores generic design strategies for the construction of FRET-based sensor proteins and other protein switches. In the present article, I show that designing FRET sensors based on mutually exclusive domain interactions provides a robust method to engineer sensors with predictable properties and an inherently large change in emission ratio.

Robust red FRET sensors using self-associating fluorescent domains.

Elucidation of subcellular signaling networks by multiparameter imaging is hindered by a lack of sensitive FRET pairs spectrally compatible with the classic CFP/YFP pair. Here, we present a generic strategy to enhance the traditionally poor sensitivity of red FRET sensors by developing self-associating variants of mOrange and mCherry that allow sensors to switch between well-defined on- and off states. Requiring just a single mutation of the mFruit domain, this new FRET pair improved the dynamic range of protease sensors up to 10-fold and was essential to generate functional red variants of CFP-YFP-based Zn(2+) sensors.

Colorful calcium sensors.

(R)evolution of protein-based calcium sensors: Expanding the toolbox of genetically encoded calcium sensors with new colors and traits is important for understanding calcium signaling and its relation to other intracellular pathways. Campbell and co-workers have used a new directed-evolution strategy to develop a rich palette of new sensors, including the first red-shifted, genetically encoded calcium sensor.

Oxidative stress of Burkholderia cenocepacia induces insertion sequence-mediated genomic rearrangements that interfere with macrorestriction-based genotyping.

Burkholderia cenocepacia can cause serious infections and epidemics in patients with cystic fibrosis (CF). A CF population in the Czech Republic experienced an epidemic outbreak caused by a B. cenocepacia ST-32 strain.