Scaling the Functional Nanopore (FuN) Screen: Systematic Evaluation of Self-Assembling Membrane Peptides and Extension with a K-Responsive Fluorescent Protein Sensor.

The functional analysis of protein nanopores is typically conducted in planar lipid bilayers or liposomes exploiting high-resolution but low-throughput electrical and optical read-outs. Yet, the reconstitution of protein nanopores still constitutes an empiric and low-throughput process. Addressing these limitations, nanopores can now be analyzed using the functional nanopore (FuN) screen exploiting genetically encoded fluorescent protein sensors that resolve distinct nanopore-dependent Ca in- and efflux patterns across the inner membrane of .

Dissecting the Permeability of the Cell Envelope to a Small Molecule Using Tailored Intensiometric Fluorescent Protein Sensors.

Membranes provide a highly selective barrier that defines the boundaries of any cell while providing an interface for communication and nutrient uptake. However, despite their central physiological role, our capacity to study or even engineer the permeation of distinct solutes across biological membranes remains rudimentary. This especially applies to Gram-negative bacteria, where the outer and inner membrane impose two permeation barriers.

iFLinkC-X: A Scalable Framework to Assemble Bespoke Genetically Encoded Co-polymeric Linkers of Variable Lengths and Amino Acid Composition.

Linker engineering is rapidly gaining prominence as protein engineers and synthetic biologists construct increasingly sophisticated protein assemblies capable of executing complex molecular functions in the context of biosensing, biocatalysis, or biotherapeutics. Depending on the application, the structural and functional requirements imposed on the underlying linkers can differ vastly. At the same time, there is a distinct lack of methods to effectively code linkers at the level of DNA and tailor them to the functional requirements of different fusion proteins.

Functional Nanopore Screen: A Versatile High-Throughput Assay to Study and Engineer Protein Nanopores in .

Nanopores comprise a versatile class of membrane proteins that carry out a range of key physiological functions and are increasingly developed for different biotechnological applications. Yet, a capacity to study and engineer protein nanopores by combinatorial means has so far been hampered by a lack of suitable assays that combine sufficient experimental resolution with throughput. Addressing this technological gap, the functional nanopore (FuN) screen now provides a quantitative and dynamic readout of nanopore assembly and function in the context of the inner membrane of .

Ultrasensitive and Selective Protein Recognition with Nanobody-Functionalized Synthetic Nanopores.

The development of flexible and reconfigurable sensors that can be readily tailored toward different molecular analytes constitutes a key goal and formidable challenge in biosensing. In this regard, synthetic nanopores have emerged as potent physical transducers to convert molecular interactions into electrical signals. Yet, systematic strategies to functionalize their surfaces with receptor proteins for the selective detection of molecular analytes remain scarce.

Synthetic protein switches: Combinatorial linker engineering with iFLinkC.

Linker engineering constitutes a critical, yet frequently underestimated aspect in the construction of synthetic protein switches and sensors. Notably, systematic strategies to engineer linkers by predictive means remain largely elusive to date. This is primarily due to our insufficient understanding how the biophysical properties that underlie linker functions mediate the conformational transitions in artificially engineered protein switches and sensors.

Linker Engineering in the Context of Synthetic Protein Switches and Sensors.

Linkers play critical roles in the construction of synthetic protein switches and sensors as they functionally couple a receptor with an actuator. With an increasing number of molecular toolboxes and experimental strategies becoming available that can be applied to engineer protein switches and sensors with tailored response functions, optimising the connecting linkers remains an idiosyncratic and empiric process. This review aims to provide an in-depth analysis of linker motifs, the biophysical properties they confer, and how they impact the performance of synthetic protein switches and sensors while identifying trends, mechanisms, and strategies that underlie the most potent switches and sensors.

Ultrasensitive and Selective Copper(II) Detection: Introducing a Bioinspired and Robust Sensor.

A nanopore-based Cu -sensing system is reported that allows for an ultrasensitive and selective detection of Cu with the possibility for a broad range of applications, for example in medical diagnostics. A fluorescent ATCUN-like peptide 5/6-FAM-Dap-β-Ala-His is employed to selectively bind Cu ions in the presence of Ni and Zn and was crafted into ion track-etched nanopores. Upon Cu binding the fluorescence of the peptide sensor is quenched, permitting the detection of Cu in solution.

iFLinkC: an iterative functional linker cloning strategy for the combinatorial assembly and recombination of linker peptides with functional domains.

Recent years have witnessed increasing efforts to engineer artificial biological functions through recombination of modular-organized toolboxes of protein scaffolds and parts. A critical, yet frequently neglected aspect concerns the identity of peptide linkers or spacers connecting individual domains which remain poorly understood and challenging to assemble. Addressing these limitations, iFlinkC comprises a highly scalable DNA assembly process that facilitates the combinatorial recombination of functional domains with linkers of varying length and flexibility, thereby overcoming challenges with high GC-content and the repeat nature of linker elements.

Engineering artificial signalling functions with proteases.

Proteases have emerged as a promising class of enzymes to build post-translationally regulated signalling functions in diverse organisms and cell types ranging from simple prokaryotes to higher eukaryotes and in reconstituted systems in vitro. An expanding repertoire of proteases can now be readily configured to build tailored sensors, switches and transducers, and is increasingly facilitating the construction of complex sensory systems for a variety of biotechnological and biomedical applications. This is complemented by an increasing understanding of the fundamental design principles underlying biological signal processing at both protein-level and circuit-level that is now actively probed through synthesis.

Photolithographic Fabrication of Micro Apertures in Dry Film Polymer Sheets for Channel Recordings in Planar Lipid Bilayers.

Planar lipid bilayers constitute a versatile method for measuring the activity of protein channels and pores on a single molecule level. Ongoing efforts attempt to tailor this method for detecting biomedically relevant target analytes or for high-throughput screening of drugs. To improve the mechanical stability of bilayer recordings, we use a thin-film epoxy resist ADEX as septum in free-standing vertical bilayers.

Engineering and Characterizing Synthetic Protease Sensors and Switches.

Proteases are finding an increasing number of applications as molecular tools and reporters in biotechnology and basic research. Proteases are also increasingly incorporated into synthetic genetic signaling circuits equipping cells with tailored new functions. In the majority of cases however, proteases are employed in constitutively active forms which limits their utility and application as molecular sensors.

Synthetic Protein Switches: Theoretical and Experimental Considerations.

Synthetic protein switches with tailored response functions are finding increasing applications as tools in basic research and biotechnology. With a number of successful design strategies emerging, the construction of synthetic protein switches still frequently necessitates an integrated approach that combines detailed biochemical and biophysical characterization in combination with high-throughput screening to construct tailored synthetic protein switches. This is increasingly complemented by computational strategies that aim to reduce the need for costly empirical optimization and thus facilitate the protein design process.

Ultrasensitive Scaffold-Dependent Protease Sensors with Large Dynamic Range.

The rational construction of synthetic protein switches with predefined input-output parameters constitutes a key goal of synthetic biology with many potential applications ranging from metabolic engineering to diagnostics. Yet, generally applicable strategies to construct tailor-engineered protein switches have so far remained elusive. Here, we use SpyTag/SpyCatcher-mediated protein ligation to engineer modularly organized, scaffold-dependent protease sensors that exploit a combination of affinity targeting and protease-inducible protein-protein interactions.

Engineered PQQ-Glucose Dehydrogenase as a Universal Biosensor Platform.

Biosensors with direct electron output hold promise for nearly seamless integration with portable electronic devices. However, so far, they have been based on naturally occurring enzymes that significantly limit the spectrum of detectable analytes. Here, we present a novel biosensor architecture based on analyte-driven intermolecular recombination and activity reconstitution of a re-engineered component of glucometers: PQQ-glucose dehydrogenase.

Engineering PQQ-glucose dehydrogenase into an allosteric electrochemical Ca(2+) sensor.

Electrochemical biosensors convert biological events to an electrical current. To date most electrochemical biosensors exploit activities of naturally occurring enzymes. Here we demonstrated that insertion of a calmodulin domain into the redox enzyme PQQ-glucose dehydrogenase resulted in a selective Ca(2+) biosensor that could be used to rapidly measure Ca(2+) concentrations in human biological fluids.

Semisynthetic tRNA complement mediates in vitro protein synthesis.

Genetic code expansion is a key objective of synthetic biology and protein engineering. Most efforts in this direction are focused on reassigning termination or decoding quadruplet codons. While the redundancy of genetic code provides a large number of potentially reassignable codons, their utility is diminished by the inevitable interaction with cognate aminoacyl-tRNAs.

Towards the systematic mapping and engineering of the protein prenylation machinery in Saccharomyces cerevisiae.

Protein prenylation is a widespread and highly conserved eukaryotic post-translational modification that endows proteins with the ability to reversibly attach to intracellular membranes. The dynamic interaction of prenylated proteins with intracellular membranes is essential for their signalling functions and is frequently deregulated in disease processes such as cancer. As a result, protein prenylation has been pharmacologically targeted by numerous drug discovery programs, albeit with limited success.

Synthetic protein switches: design principles and applications.

Protein switches are ubiquitous in biological signal transduction systems, enabling cells to sense and respond to a variety of molecular queues in a rapid, specific, and integrated fashion. Analogously, tailor-engineered protein switches with custom input and output functions have become invaluable research tools for reporting on distinct physiological states and actuating molecular functions in real time and in situ. Here, we analyze recent progress in constructing protein-based switches while assessing their potential in the assembly of defined signaling motifs.

Protease-based synthetic sensing and signal amplification.

The bottom-up design of protein-based signaling networks is a key goal of synthetic biology; yet, it remains elusive due to our inability to tailor-make signal transducers and receptors that can be readily compiled into defined signaling networks. Here, we report a generic approach for the construction of protein-based molecular switches based on artficially autoinhibited proteases. Using structure-guided design and directed protein evolution, we created signal transducers based on artificially autoinhibited proteases that can be activated following site-specific proteolysis and also demonstrate the modular design of an allosterically regulated protease receptor following recombination with an affinity clamp peptide receptor.

Assembling linear DNA templates for in vitro transcription and translation.

Cell-free expression systems provide straightforward access from genes to the corresponding proteins, involving fewer handling steps than in vivo procedures. A quick procedure to assemble a gene of interest into a linear DNA template together with 3'- and 5'-untranslated regions using a coupled uracil-excision-ligation strategy based on USER Enzyme and T4 DNA ligase. This methodology will be useful for repeated cycles of expression and in vitro selection, in which gene libraries are repeatedly assembled and their products and templates regenerated.

SNAP dendrimers: multivalent protein display on dendrimer-like DNA for directed evolution.

Display systems connect a protein with the DNA encoding it. Such systems (e.g.

Isothermal DNA amplification using the T4 replisome: circular nicking endonuclease-dependent amplification and primase-based whole-genome amplification.

In vitro reconstitution of the bacteriophage T4 replication machinery provides a novel system for fast and processive isothermal DNA amplification. We have characterized this system in two formats: (i) in circular nicking endonuclease-dependent amplification (cNDA), the T4 replisome is supplemented with a nicking endonuclease (Nb.BbvCI) and a reverse primer to generate a well-defined uniform double-stranded linear product and to achieve up to 1100-fold linear amplification of a plasmid in 1 h.

An efficient method to assemble linear DNA templates for in vitro screening and selection systems.

A method is presented to assemble a gene of interest into a linear DNA template with all the components necessary for in vitro transcription and translation in approximately 90 min. Assembly is achieved using a coupled uracil excision-ligation strategy based on USER Enzyme and T4 DNA ligase, which allows the simultaneous and seamless assembly of three different PCR products. The method is suitable for screening and selection systems of very high throughput as up to 10(11) molecules can be efficiently assembled and purified in reaction volumes of 100 microl.

SPORCalc: A development of a database analysis that provides putative metabolic enzyme reactions for ligand-based drug design.

Understanding both the enzyme reactions that contribute to intermediate metabolism and the biochemical fate of candidate therapeutic and toxic agents are essential for drug design. Traditional metabolic databases indicate whether reactions have been observed but do not provide the likelihoods of reactions occurring, for example those of mixed function oxygenases and oxidases, during phase I metabolism. The desire for more quantitative predictions motivated the development of the recently introduced Substrate Product Occurrence Ratio Calculator (SPORCalc) that identifies metabolically labile atom positions in candidate compounds.