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 in vitro 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 Ca2+ in- and efflux patterns across the inner membrane of Escherichia coli. With a primary proof-of-concept established for the S2168 holin, and thereof based recombinant nanopore assemblies, the question arises to what extent alternative nanopores can be analyzed with the FuN screen and to what extent alternative fluorescent protein sensors can be adapted. Focusing on self-assembling membrane peptides, three sets of 13 different nanopores are assessed for their capacity to form nanopores in the context of the FuN screen. Nanopores tested comprise both natural and computationally designed nanopores. Further, the FuN screen is extended to K+-specific fluorescent protein sensors and now provides a capacity to assess the specificity of a nanopore or ion channel. Finally, a comparison to high-resolution biophysical and electrophysiological studies in planar lipid bilayers provides an experimental benchmark for future studies.

Dissecting the Permeability of the Escherichia coli 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. Addressing this analytical challenge, we exemplify how the permeability of the Escherichia coli cell envelope can be dissected using a small-molecule-responsive fluorescent protein sensor. The approach is exemplified for the biotechnologically relevant macrolide rapamycin, for which we first construct an intensiometric rapamycin detector (iRapTor) while comprehensively probing key design principles in the iRapTor scaffold. Specifically, this includes the scope of minimal copolymeric linkers as a function of topology and the concomitant need for gate post residues. In a subsequent step, we apply iRapTors to assess the permeability of the E. coli cell envelope to rapamycin. Despite its lipophilic character, rapamycin does not readily diffuse across the E. coli envelope but can be enhanced by recombinantly expressing a nanopore in the outer membrane. Our study thus provides a blueprint for studying and actuating the permeation of small molecules across the prokaryotic cell envelope.

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. Addressing these limitations, a scalable framework is presented to compose co-polymeric linkers of variable lengths and amino acid composition based on a limited number of linker fragments stored in sequence-verified entry plasmids. The assembly process is exemplified for Pro-rich linkers in the context of a Zn2+-responsive dual-readout BRET/FRET sensor while examining how linker composition impacts key functional properties such as ligand affinity, dynamic range, and their ability to separate structurally distinct domains.

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

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 Escherichia coli. The assay is based on genetically encoded fluorescent protein sensors that resolve the nanopore-dependent influx of Ca2+ across the inner membrane of E. coli. Illustrating its versatile capacity, the FuN screen is first applied to dissect the molecular features that underlie the assembly and stability of nanopores formed by the S2168 holin. In a subsequent step, nanopores are engineered by recombining the transmembrane module of S2168 with different ring-shaped oligomeric protein structures that feature defined hexa-, hepta-, and octameric geometries. Library screening highlights substantial plasticity in the ability of the S2168 transmembrane module to oligomerize in alternative geometries, while the functional properties of the resultant nanopores can be fine-tuned through the identity of the connecting linkers. Overall, the FuN screen is anticipated to facilitate both fundamental studies and complex nanopore engineering endeavors with many potential applications in biomedicine, biotechnology, and synthetic biology.

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. Addressing these limitations, a general strategy is presented to immobilize nanobodies in a directional fashion onto the surface of track-etched nanopores exploiting copper-free click reactions and site-specific protein conjugation systems. The functional immobilization of three different nanobodies is demonstrated in ligand binding experiments with green fluorescent protein, mCherry, and α-amylase (α-Amy) serving as molecular analytes. Ligand binding is resolved using a combination of optical and electrical recordings displaying quantitative dose-response curves. Furthermore, a change in surface charge density is identified as the predominant molecular factor that underlies quantitative dose-responses for the three different protein analytes in nanoconfined geometries. The devised strategy should pave the way for the systematic functionalization of nanopore surfaces with biological receptors and their ability to detect a variety of analytes for diagnostic purposes.

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. The construction of synthetic protein switches and sensors therefore heavily relies on experimental trial-and-error. Yet, methods for effectively generating linker diversity at the genetic level are scarce. Addressing this technical shortcoming, iterative functional linker cloning (iFLinkC) enables the combinatorial assembly of linker elements with functional domains from sequence verified repositories that are developed and stored in-house. The assembly process is highly scalable and given its recursive nature generates linker diversity in a combinatorial and exponential fashion based on a limited number of linker elements.

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 CuII -sensing system is reported that allows for an ultrasensitive and selective detection of CuII with the possibility for a broad range of applications, for example in medical diagnostics. A fluorescent ATCUN-like peptide 5/6-FAM-Dap-ß-Ala-His is employed to selectively bind CuII ions in the presence of NiII and ZnII and was crafted into ion track-etched nanopores. Upon CuII binding the fluorescence of the peptide sensor is quenched, permitting the detection of CuII in solution. The ion transport characteristics of peptide-modified nanopore are shown to be extremely sensitive and selective towards CuII allowing to sense femtomolar CuII concentrations in human urine mimics. Washing with EDTA fully restores the CuII -binding properties of the sensor, enabling multiple repetitive measurements. The robustness of the system clearly has the potential to be further developed into an easy-to-use, lab-on-chip CuII -sensing device, which will be of great importance for bedside diagnosis and monitor of CuII levels in patients with copper-dysfunctional homeostasis.

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. The capacity of iFLinkC is demonstrated in the construction of synthetic protease switches featuring PDZ-FN3-based affinity clamps and single-chain FKBP12-FRB receptors as allosteric inputs. Library screening experiments demonstrate that linker space is highly plastic as the induction of allosterically regulated protease switches can vary from >150-fold switch-ON to >13-fold switch-OFF solely depending on the identity of the connecting linkers and relative orientation of functional domains. In addition, Pro-rich linkers yield the most potent switches contradicting the conventional use of flexible Gly-Ser linkers. Given the ease and efficiency how functional domains can be readily recombined with any type of linker, iFLinkC is anticipated to be widely applicable to the assembly of any type of fusion protein.

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. This review thus aims to summarize and analyse the most promising conceptual and experimental approaches that can be applied to build artificial signalling functions with proteases while highlighting advances, drawbacks and limitations.

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. Defined apertures with diameters between 30 µm and 100 µm were micro-fabricated by photolithography. The performance of these septa was tested by functional reconstitution of the K+ channel KcvNTS in lipid bilayers spanned over apertures in ADEX or Teflon films; the latter is conventionally used in bilayer recordings and serves as reference. We observe that the functional properties of the K+ channel are identical in both materials while ADEX provides no advantage in terms of capacitance and signal-to-noise ratio. In contrast to Teflon, however, ADEX enables long-term experimental recordings while the stability of the lipid bilayer is not compromised by pipetting solutions in and out of the recording chamber. Combined with the fact that the ADEX films can be cleaned with acetone, our results suggest that ADEX carries great potential for multiplexing bilayer chambers in robust and reusable sensing devices.

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. We use this architecture to create a suite of integrated signal sensing and amplification circuits that can detect the activity of α-thrombin and prostate specific antigen with a dynamic range covering 5 orders of magnitude. We determine the key design features critical for signal transmission between protease-based sensors, transducers, and actuators.

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. Successful computational design approaches range from analyzing phylogenetic data to infer useful structural, biophysical, and biochemical information to modeling the structure and function of proteins ab initio. The following chapter provides an overview over the theoretical considerations and experimental approaches that have been successful applied in the construction of synthetic protein switches.

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. The following chapter provides a detailed experimental protocol for converting constitutively active proteases into regulated protease receptors. Such receptors can potentially sense, transduce, and amplify any molecular input, thereby opening up a range of new applications in basic research, biotechnology, and synthetic biology.

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. We demonstrate that this sensor architecture can be rapidly adopted for the detection of immunosuppressant drugs, α-amylase protein, or protease activity of thrombin and Factor Xa. The biosensors could be stored in dried form without appreciable loss of activity. We further show that ligand-induced activity of the developed biosensors could be directly monitored by chronoamperometry, enabling construction of disposable sensory electrodes. We expect that this architecture could be expanded to the detection of other biochemical activities, post-translational modifications, nucleic acids, and inorganic molecules.

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. We were able to convert a point-of-care glucometer into Ca(2+) monitor by refurbishing it with the developed biosensor. We propose that similar engineering strategies may be used to create highly specific electrochemical biosensors to other analytes. Compatibility with cheap and ubiquitous amperometric detectors is expected to accelerate progression of these biosensors into clinical applications.

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. To address this problem, we sought to establish an in vitro protein synthesis system with a simplified synthetic tRNA complement, thereby orthogonalizing some of the sense codons. This quantitative in vitro peptide synthesis assay allowed us to analyze the ability of synthetic tRNAs to decode all of 61 sense codons. We observed that, with the exception of isoacceptors for Asn, Glu, and Ile, the majority of 48 synthetic Escherichia coli tRNAs could support protein translation in the cell-free system. We purified to homogeneity functional Asn, Glu, and Ile tRNAs from the native E. coli tRNA mixture, and by combining them with synthetic tRNAs, we formulated a semisynthetic tRNA complement for all 20 amino acids. We further demonstrated that this tRNA complement could restore the protein translation activity of tRNA-depleted E. coli lysate to a level comparable to that of total native tRNA. To confirm that the developed system could efficiently synthesize long polypeptides, we expressed three different sequences coding for superfolder GFP. This novel semisynthetic translation system is a powerful tool for tRNA engineering and potentially enables the reassignment of at least 9 sense codons coding for Ser, Arg, Leu, Pro, Thr, and Gly.

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. To a large extent, this can be attributed to an insufficient understanding of the interplay of different protein prenyltransferases and the combinatorial diversity of the prenylatable sequence space. Here, we report a high-throughput, growth-based genetic selection assay in Saccharomyces cerevisiae based on the Ras Recruitment System which, for the first time, has allowed us to create a comprehensive map of prenylatable protein sequences in S. cerevisiae. We demonstrate that potential prenylatable space is sparsely (6.2%) occupied leaving room for creation of synthetic orthogonal prenylatable sequences. To experimentally demonstrate that, we used the developed platform to engineer mutant farnesyltransferases that efficiently prenylate substrate motives that are not recognised by endogenous protein prenyltransferases. These uncoupled mutants can now be used as starting points for the systematic engineering of the eukaryotic protein prenylation machinery.

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. We anticipate such systems will ultimately pave the way towards a new generation of molecular diagnostics and facilitate the construction of artificial signaling systems that operate in parallel to the signaling machinery of a host cell for applications in synthetic biology.

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. Notably, the receptor's mode of action can be varied from >5-fold switch-OFF to >30-fold switch-ON solely by changing the length of the connecting linkers, demonstrating a high functional plasticity not previously observed in naturally occurring receptor systems. We also create an integrated signaling circuit based on two orthogonal autoinhibited protease units that can propagate and amplify molecular queues generated by the protease receptor. Finally, we present a generic two-component receptor architecture based on proximity-based activation of two autoinhibited proteases. Overall, the approach allows the design of protease-based signaling networks that, in principle, can be connected to any biological process.