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.

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.

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.

Diagnostic reliability of the Berlin classification for complex MCA aneurysms-usability in a series of only giant aneurysms.

BACKGROUND AND OBJECTIVE: The main challenge of bypass surgery of complex MCA aneurysms is not the selection of the bypass type but the initial decision-making of how to exclude the affected vessel segment from circulation. To this end, we have previously proposed a classification for complex MCA aneurysms based on the preoperative angiography. The current study aimed to validate this new classification and assess its diagnostic reliability using the giant aneurysm registry as an independent data set. METHODS: We reviewed the pretreatment neuroimaging of 51 patients with giant (> 2.5 cm) MCA aneurysms from 18 centers, prospectively entered into the international giant aneurysm registry. We classified the aneurysms according to our previously proposed Berlin classification for complex MCA aneurysms. To test for interrater diagnostic reliability, the data set was reviewed by four independent observers. RESULTS: We were able to classify all 51 aneurysms according to the Berlin classification for complex MCA aneurysms. Eight percent of the aneurysm were classified as type 1a, 14% as type 1b, 14% as type 2a, 24% as type 2b, 33% as type 2c, and 8% as type 3. The interrater reliability was moderate with Fleiss's Kappa of 0.419. CONCLUSION: The recently published Berlin classification for complex MCA aneurysms showed diagnostic reliability, independent of the observer when applied to the MCA aneurysms of the international giant aneurysm registry.

Bioluminescent detection of viral surface proteins using branched multivalent protein switches.

Fast and reliable virus diagnostics is key to prevent the spread of viruses in populations. A hallmark of viruses is the presence of multivalent surface proteins, a property that can be harnessed to control conformational switching in sensor proteins. Here, we introduce a new sensor platform (dark-LUX) for the detection of viral surface proteins consisting of a general bioluminescent framework that can be post-translationally functionalized with separately expressed binding domains. The platform relies on (1) plug-and-play bioconjugation of different binding proteins via SpyTag/SpyCatcher technology to create branched protein structures, (2) an optimized turn-on bioluminescent switch based on complementation of the split-luciferase NanoBiT upon target binding and (3) straightforward exploration of the protein linker space. The influenza A virus (IAV) surface proteins hemagglutinin (HA) and neuraminidase (NA) were used as relevant multivalent targets to establish proof of principle and optimize relevant parameters such as linker properties, choice of target binding domains and the optimal combination of the competing NanoBiT components SmBiT and DarkBiT. The sensor framework allows rapid conjugation and exchange of various binding domains including scFvs, nanobodies and de novo designed binders for a variety of targets, including the construction of a heterobivalent switch that targets the head and stem region of hemagglutinin. The modularity of the platform thus allows straightforward optimization of binding domains and scaffold properties for existing viral targets, and is well suited to quickly adapt bioluminescent sensor proteins to effectively detect newly evolving viral epitopes.

Bioluminescence Goes Dark: Boosting the Performance of Bioluminescent Sensor Proteins Using Complementation Inhibitors.

Bioluminescent sensor proteins have recently gained popularity in both basic research and point-of-care diagnostics. Sensor proteins based on intramolecular complementation of split NanoLuc are particularly attractive because their intrinsic modular design enables for systematic tuning of sensor properties. Here we show how the sensitivity of these sensors can be enhanced by the introduction of catalytically inactive variants of the small SmBiT subunit (DarkBiTs) as intramolecular inhibitors. Starting from previously developed bioluminescent antibody sensor proteins (LUMABS), we developed single component, biomolecular switches with a strongly reduced background signal for the detection of three clinically relevant antibodies, anti-HIV1-p17, cetuximab (CTX), and an RSV neutralizing antibody (101F). These new dark-LUMABS sensors showed 5-13-fold increases in sensitivity which translated into lower limits of detection. The use of DarkBiTs as competitive intramolecular inhibitor domains is not limited to the LUMABS sensor family and might be used to boost the performance of other bioluminescent sensor proteins based on split luciferase complementation.

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.

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.

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.

An interdisciplinary approach to the treatment of a complex infraclinoidal internal carotid artery aneurysm.

We present the case of a 49-year-old woman with sudden onset of severe headaches and a ruptured aneurysm located inside the fenestration of the infraclinoid part of the internal carotid artery in the segments C4 and C5 distal to the origin of the ophthalmic artery. An interdisciplinary approach enabled the successful treatment of the aneurysm by wrapping and stent-assisted coiling. We discuss this rare congenital anomaly of a fenestrated internal carotid artery together with the 12 other cases published worldwide.

A paper-based, cell-free biosensor system for the detection of heavy metals and date rape drugs.

Biosensors have emerged as a valuable tool with high specificity and sensitivity for fast and reliable detection of hazardous substances in drinking water. Numerous substances have been addressed using synthetic biology approaches. However, many proposed biosensors are based on living, genetically modified organisms and are therefore limited in shelf life, usability and biosafety. We addressed these issues by the construction of an extensible, cell-free biosensor. Storage is possible through freeze drying on paper. Following the addition of an aqueous sample, a highly efficient cell-free protein synthesis (CFPS) reaction is initiated. Specific allosteric transcription factors modulate the expression of 'superfolder' green fluorescent protein (sfGFP) depending on the presence of the substance of interest. The resulting fluorescence intensities are analyzed with a conventional smartphone accompanied by simple and cheap light filters. An ordinary differential equitation (ODE) model of the biosensors was developed, which enabled prediction and optimization of performance. With an optimized cell-free biosensor based on the Shigella flexneri MerR transcriptional activator, detection of 6 µg/L Hg(II) ions in water was achieved. Furthermore, a completely new biosensor for the detection of gamma-hydroxybutyrate (GHB), a substance used as date-rape drug, was established by employing the naturally occurring transcriptional repressor BlcR from Agrobacterium tumefaciens.

Membrane Protein Production in E. coli Lysates in Presence of Preassembled Nanodiscs.

Cell-free expression allows to synthesize membrane proteins in completely new formats that can relatively easily be customized for particular applications. Amphiphilic superstructures such as micelles, lipomicelles, or nanodiscs can be provided as nano-devices for the solubilization of membrane proteins. Defined empty bilayers in the form of nanodiscs offer native like environments for membrane proteins, supporting functional folding, proper oligomeric assembly as well as stability. Even very difficult and detergent-sensitive membrane proteins can be addressed by the combination of nanodisc technology with efficient cell-free expression systems as the direct co-translational insertion of nascent membrane proteins into supplied preassembled nanodiscs is possible. This chapter provides updated protocols for the synthesis of membrane proteins in presence of preassembled nanodiscs suitable for emerging applications such as screening of lipid effects on membrane protein function and the modulation of oligomeric complex formation.

The dilemma of complicated shunt valves: How to identify patients with posthemorrhagic hydrocephalus after aneurysmatic subarachnoid hemorrhage who will benefit from a simple valve?

BACKGROUND: Sophisticated shunt valves provide the possibility of pressure adjustment and antisiphon control but have a higher probability of valve dysfunction especially in a posthemorrhagic setting. The aim of the present study is to analyze the clinical outcome of patients with shunt dependent posthemorrhagic hydrocephalus after aneurysmatic subarachnoid hemorrhage (SAH) in order to identify patients who would benefit from a simple differential pressure valve. METHODS: From 2000 to 2013, 547 patients with aneurysmatic SAH were treated at our institution, 114 underwent ventricular shunt placement (21.1%). 47 patients with available pre- and post-operative computed tomography scans, and an available follow-up of minimum 6 months were included. In order to measure the survival time which a nonprogrammable differential pressure valve would have had in an individual patient we defined the initial equalized shunt survival time (IESS). IESS is the time until surgical revisions of fixed differential pressure or flow-regulated valves for the treatment of over- or under-drainage as well as re-programming of adjustable valves due to over- or under-drainage. RESULTS: Twenty patients were treated with fixed differential pressure valves, 15 patients were treated with flow-regulated valves, and 12 underwent ventriculoperitoneal (VP) shunt placement with differential pressure valves assisted by a gravitational unit. Patients who reacted with remarkable changes of the ventricular width after the insertion of external ventricular drainage (EVD), before shunt placement, showed a significantly longer IESS. CONCLUSIONS: Decline of the ventricular width after EVD placement was a predictor for successful VP shunt therapy in the later course of disease. Possibly, this could allow identifying patients who benefit from a simple differential pressure valve or a flow-regulated valve, and thus could possibly avoid valve-associated complications of a programmable valve in the later course of disease.