Thermostable WW-Domain Scaffold to Design Functional ß-Sheet Miniproteins.

There has been a recent surge in the design of miniproteins for medicinal chemistry, biomaterial design, or synthetic biology. In particular, there is an interest in peptide scaffolds that fold reliably, predictably, and with solid stability. In this article, we present the design of a highly thermostable WW domain, a three-stranded ß-sheet motif, with a superior melting temperature of about 90 °C to serve as a core scaffold onto which receptor-like properties can be grafted. We have performed specific rounds of sequence iteration on a WW-domain consensus sequence to decipher sequence positions that affect structural and, thus, thermal stability. We identified a sequence-structure relationship that yields a highly thermostable WW-domain scaffold. High-resolution NMR spectroscopy was applied, which enabled the identification of structural features at the atomic scale that contribute to this high thermostability. Finally, we grafted the binding motifs of the three WW-domain groups─Group I, Group II/III, and Group IV─and organophosphate and metal binding onto the highly thermostable WW-domain scaffold and obtained thermostable de novo WW domains that indeed display the different binding modes that were intended. The organophosphate-binding WW domains exhibit melting temperatures that are up to 34 K higher than previously reported top-down designs. These results impressively demonstrate that the highly thermostable WW-domain core scaffold is a solid platform for the design of discrete and reliably folding functional ß-sheet peptide miniproteins, providing an essential addition to the toolbox of peptide scaffolds previously used in synthetic biology and material design.

Peptide Boronic Acids by Late-Stage Hydroboration on the Solid Phase.

Organoboron compounds have a wide range of applications in numerous research fields, and metmhods to incorporate them in biomolecules are much sought after. Here, on-resin chemical syntheses of aliphatic and vinylogous peptide boronic acids are presented by transition metal-catalyzed late-stage hydroboration of alkene and alkyne groups in peptides and peptoids, for example on allyl- and propargylglycine residues, using readily available chemicals. These methods yield peptide boronic acids with much shorter linkers than previously reported on-resin methods. Furthermore, the methods are regio- and stereoselective, compatible with all canonical amino acid residues and can be applied to short, long, and in part even "difficult" peptide sequences. In a feasibility study, the protected peptide vinylboronic acids are further derivatized by the Petasis reaction using salicylaldehyde derivatives. The ability of the obtained peptide boronic acids to reversibly bind to carbohydrates is demonstrated in a catch-release model experiment using a fluorescently labeled peptide boronic acid on cross-linked dextran beads. In summary, this highlights the potential of the target compounds for drug discovery, glycan-specific target recognition, controlled release, and diagnostics.

Multimaterial 3D Laser Printing of Cell-Adhesive and Cell-Repellent Hydrogels.

Here, a straightforward method is reported for manufacturing 3D microstructured cell-adhesive and cell-repellent multimaterials using two-photon laser printing. Compared to existing strategies, this approach offers bottom-up molecular control, high customizability, and rapid and precise 3D fabrication. The printable cell-adhesive polyethylene glycol (PEG) based material includes an Arg-Gly-Asp (RGD) containing peptide synthesized through solid-phase peptide synthesis, allowing for precise control of the peptide design. Remarkably, minimal amounts of RGD peptide (< 0.1 wt%) suffice for imparting cell-adhesiveness, while maintaining identical mechanical properties in the 3D printed microstructures to those of the cell-repellent, PEG-based material. Fluorescent labeling of the RGD peptide facilitates visualization of its presence in cell-adhesive areas. To demonstrate the broad applicability of the system, the fabrication of cell-adhesive 2.5D and 3D structures is shown, fostering the adhesion of fibroblast cells within these architectures. Thus, this approach allows for the printing of high-resolution, true 3D structures suitable for diverse applications, including cellular studies in complex environments.

Design of Functional Globular ß-Sheet Miniproteins.

The design of discrete ß-sheet peptides is far less advanced than e. g. the design of α-helical peptides. The reputation of ß-sheet peptides as being poorly soluble and aggregation-prone often hinders active design efforts. Here, we show that this reputation is unfounded. We demonstrate this by looking at the ß-hairpin and WW domain. Their structure and folding have been extensively studied and they have long served as model systems to investigate protein folding and folding kinetics. The resulting fundamental understanding has led to the development of hyperstable ß-sheet scaffolds that fold at temperatures of 100 °C or high concentrations of denaturants. These have been used to design functional miniproteins with protein or nucleic acid binding properties, in some cases with such success that medical applications are conceivable. The ß-sheet scaffolds are not always completely rigid, but can be specifically designed to respond to changes in pH, redox potential or presence of metal ions. Some engineered ß-sheet peptides also exhibit catalytic properties, although not comparable to those of natural proteins. Previous reviews have focused on the design of stably folded and non-aggregating ß-sheet sequences. In our review, we now also address design strategies to obtain functional miniproteins from ß-sheet folding motifs.

Relationship of Thermostability and Binding Affinity in Metal-binding WW-Domain Minireceptors.

The design of metallo-miniproteins advances our understanding of the structural and functional roles of metals in proteins. We recently designed a metal-binding WW domain, WW-CA-Nle, which displays three histidine residues on its surface for coordination of divalent metals Ni(II), Zn(II) and Cu(II). However, WW-CA-Nle is a molten globule in the apo state and thus showed only moderate binding affinities with Kd values in the µM regime. In this report, we hypothesize that improved thermal stability of the apo state of the metal binding WW-domain scaffold should lead to improved preorganization of the metal-binding site and consequently to higher metal-binding affinities. By redesigning WW-CA-Nle, we obtained WW-CA variants, WW-CA-min and WW-CA-ANG, which were fully folded in the apo states and displayed moderate to excellent thermostabilities in the apo and holo states. We were able to show that the improved thermal stabilities led to improved metal binding, which was reflected in Kd values that were at least one order of magnitude lower compared to WW-CA-Nle. EPR spectroscopy and ITC measurements revealed a better defined and predisposed metal binding site in WW-CA-ANG.

Rapid On-Resin N-Formylation of Peptides as One-Pot Reaction.

N-formylation is a common pre- and post-translational modification of the N-terminus or the lysine side chain of peptides and proteins that plays a role in the initiation of immune responses, gene expression, or epigenetics. Despite its high biological relevance, protocols for the chemical N-formylation of synthetic peptides are scarce. The few available methods are elaborate in their execution and the yields are highly sequence-dependent. We present a rapid, easy-to-use one-pot procedure that runs at room temperature and can be used to formylate protected peptides at both the N-terminus and the lysine side chain on the resin in near-quantitative yields. Only insensitive, storage-stable standard chemicals - formic acid, acetic anhydride, pyridine and DMF - are used. Formylation works for both short and long peptides of up to 34 amino acids and over the spectrum of canonical amino acids.

HAV-Peptides Attached to Colloidal Probes Faithfully Detect E-Cadherins Displayed on Living Cells.

Cell adhesion molecules are crucial for a variety of biological processes, including wound healing, barrier formation and tissue homeostasis. One of them is E-cadherin which is generally found at adherent junctions between epithelial cells. To identify this molecule on the surface of cells, E-cadherin mimetic peptides with a critical amino acid sequence of HAV (histidine-alanine-valine) were synthesized and attached to solid-supported membranes covering colloidal probes. Two different functionalization strategies were established, one based on the complexation of DOGS-NTA(Ni) with a polyhistidine-tagged HAV-peptide and the other one relying on the formation of a HAV-lipopeptide using in situ maleimide-thiol coupling. Binding studies were performed to verify the ability of the peptides to attach to the membrane surface. Compared to the non-covalent attachment via the His-tag, we achieved a higher yield by lipopeptide formation. Colloidal probes functionalized with HAV-peptides were employed to measure the presence of E-cadherins on living cells either using video particle tracking or force spectroscopy. Here, human HaCaT cells were examined confirming the specific interaction of the HAV-peptide with the E-cadherin of the cells. Statistical methods were also used to determine the number of single-bond ruptures and the force of a single bond. These findings may be essential for the development of novel biosynthetic materials given their potential to become increasingly relevant in medical applications.

An Engineered ß-Hairpin Peptide Forming Thermostable Complexes with ZnII , NiII , and CuII through a His3 Site.

The three-dimensional structure of a peptide, which determines its function, can denature at elevated temperatures, in the presence of chaotropic reagents, or in organic solvents. These factors limit the applicability of peptides. Herein, we present an engineered ß-hairpin peptide containing a His3 site that forms complexes with ZnII , NiII , and CuII . Circular dichroism spectroscopy shows that the peptide-metal complexes exhibit melting temperatures up to 80 °C and remain folded in 6 M guanidine hydrochloride as well as in organic solvents. Intrinsic fluorescence titration experiments were used to determine the dissociation constants of metal binding in the nano- to sub-nanomolar range. The coordination geometry of the peptide-CuII complex was studied by EPR spectroscopy, and a distorted square planar coordination geometry with weak interactions to axial ligands was revealed. Due to their impressive stability, the presented peptide-metal complexes open up interesting fields of application, such as the development of a new class of peptide-metal catalysts for stereoselective organic synthesis or the directed design of extremophilic ß-sheet peptides.

Identification of novel functional mini-receptors by combinatorial screening of split-WW domains.

ß-Sheet motifs such as the WW domain are increasingly being explored as building blocks for synthetic biological applications. Since the sequence-structure relationships of ß-sheet motifs are generally complex compared to the well-studied α-helical coiled coil (CC), other approaches such as combinatorial screening should be included to vary the function of the peptide. In this study, we present a combinatorial approach to identify novel functional mini-proteins based on the WW-domain scaffold, which takes advantage of the successful reconstitution of the fragmented WW domain of hPin1 (hPin1WW) by CC association. Fragmentation of hPin1WW was performed in both loop 1 (CC-hPin1WW-L1) and loop 2 (CC-hPin1WW-L2), and the respective fragments were linked to the strands of an antiparallel heterodimeric CC. Structural analysis by CD and NMR spectroscopy revealed structural reconstitution of the WW-domain scaffold only in CC-hPin1WW-L1, but not in CC-hPin1WW-L2. Furthermore, by using 1H-15N HSQC NMR, fluorescence and CD spectroscopy, we demonstrated that binding properties of fragmented hPin1WW in CC-hPin1WW-L1 were fully restored by CC association. To demonstrate the power of this approach as a combinatorial screening platform, we synthesized a four-by-six library of N- and C-terminal hPin1WW-CC peptide fragments that was screened for a WW domain that preferentially binds to ATP over cAMP, phophocholine, or IP6. Using this screening platform, we identified one WW domain, which specifically binds ATP, and a phosphorylcholine-specific WW-based mini-receptor, both having binding dissociation constants in the lower micromolar range.

Late-Stage Functionalisation of Peptides on the Solid Phase by an Iodination-Substitution Approach.

The functionalisation of peptides at a late synthesis stage holds great potential, for example, for the synthesis of peptide pharmaceuticals, fluorescent biosensors or peptidomimetics. Here we describe an on-resin iodination-substitution reaction sequence on homoserine that is also suitable for peptide modification in a combinatorial format. The reaction sequence is accessible to a wide range of sulfur nucleophiles with various functional groups including boronic acids, hydroxy groups or aromatic amines. In this way, methionine-like thioethers or thioesters and thiosulfonates are accessible. Next to sulfur nucleophiles, selenols, pyridines and carboxylic acids were successfully used as nucleophiles, whereas phenols did not react. The late-stage iodination-substitution approach is not only applicable to short peptides but also to the more complex 34-amino-acid WW domains. We applied this strategy to introduce 7-mercapto-4-methylcoumarin into a switchable ZnII responsive WW domain to design an iFRET-based ZnII sensor.

The CSY-protecting group in the microwave-assisted synthesis of aggregation-prone peptides.

This report describes the application of cyanosulfurylide (CSY)-protected aspartatic acid building blocks in microwave-assisted synthesis of aggregation-prone protein domains. We present a synthesis of Fmoc-Asp(CSY)-OH on a multigram scale, as well as procedures for the microwave-assisted synthesis of CSY-protected peptides, and CSY cleavage in partially folded or aggregation-prone peptides.

Switchable Zinc(II)-Responsive Globular ß-Sheet Peptide.

The natural function of many proteins depends on their ability to switch their conformation driven by environmental changes. In this work, we present a small, monomeric ß-sheet peptide that switches between a molten globule and a folded state through Zn(II) binding. The solvent-exposed hydrophobic core on the ß-sheet surface was substituted by a His3-site, whereas the internal hydrophobic core was left intact. Zn(II) is specifically recognized by the peptide relative to other divalent metal ions, binds in the lower micromolar range, and can be removed and re-added without denaturation of the peptide. In addition, the peptide is fully pH-switchable, has a pKa of about 6, and survives several cycles of acidification and neutralization. In-depth structural characterization of the switch was achieved by concerted application of circular dichroism (CD) and multinuclear NMR spectroscopy. Thus, this study represents a viable approach toward a globular ß-sheet Zn(II) mini-receptor prototype.

Effect of Methionine Sulfoxide on the Synthesis and Purification of Aggregation-Prone Peptides.

A two-step synthesis for methionine-containing hydrophobic and/or aggregation-prone peptides is presented that takes advantage of the reversibility of methionine oxidation. The use of polar methionine sulfoxide as a building block in solid-phase peptide synthesis improves the synthesis quality and yields the crude peptide, with significantly improved solubility compared to the reduced species. This facilitates the otherwise often laborious peptide purification by high-performance liquid chromatography. The subsequent reduction proceeds quantitatively. This approach has been optimised with the methionine-rich Tar-DNA-binding protein 43 (307-347), but is also more generally applicable, as demonstrated by the syntheses of human calcitonin and two aggregation-prone peptides from the human prion protein.

The influence of prompts on final year medical students' learning process and achievement in ECG interpretation.

Objective: ECG interpretation is prone to errors that can lead to relevant misdiagnoses and incorrect treatment. Prompts are one way in lectures to encourage learning from one's own mistakes and to reduce error rates. Prompts are measures such as questions, hints, and suggestions of content-related or metacognitive nature, which can lead to self-explanation in the learner and thus to a deeper understanding of an issue. The aim of the study was therefore to investigate whether the use of prompts can reduce the error rate in ECG interpretation among students. Method: In a 2x2 experimental test and control group design, N=100 final year medical students carried out ECG interpretation tasks in the form of online case vignettes in CASUS®. In these tasks, justification prompts (B) and error analysis prompts (F) were systematically varied in four groups and the learning success was measured using a knowledge test. In addition, prior knowledge in ECG interpretation, motivation, interest in the topic, subjective confidence in ECG interpretation, and cognitive load was collected. Results: Neither error analysis prompts nor justification prompts had a significant effect on the correct ECG interpretation by students, F(1,96)=1.03, p=.31. Justification prompts seemed to have a positive effect on the confidence of answering the questions, F(1,96)=10.15, p=.002, partial η2 =.10; and a negative effect on student motivation, F(1,96)=8.13 , p=.005, partial η2 =.08; but both with comparable diagnostic accuracy. Conclusion: The present study could not confirm the positive effects of prompts on the error rate in ECG interpretation reported in the literature but showed significant effects on subjective confidence and motivation which should be investigated in further studies.

Guiding Biomolecular Interactions in Cells Using de Novo Protein-Protein Interfaces.

An improved ability to direct and control biomolecular interactions in living cells would have an impact on synthetic biology. A key issue is the need to introduce interacting components that act orthogonally to endogenous proteomes and interactomes. Here, we show that low-complexity, de novo designed protein-protein interaction (PPI) domains can substitute for natural PPIs and guide engineered protein-DNA interactions in Escherichia coli. Specifically, we use de novo homo- and heterodimeric coiled coils to reconstitute a cytoplasmic split adenylate cyclase, recruit RNA polymerase to a promoter and activate gene expression, and oligomerize both natural and designed DNA-binding domains to repress transcription. Moreover, the stabilities of the heterodimeric coiled coils can be modulated by rational design and, thus, adjust the levels of gene activation and repression in vivo. These experiments demonstrate the possibilities for using designed proteins and interactions to control biomolecular systems such as enzyme cascades and circuits in cells.

De Novo Designed α-Helical Coiled-Coil Peptides as Scaffolds for Chemical Reactions.

Coiled coils (CCs) are well-understood protein-folding motifs. They appear in a variety of oligomer states and as homo- and heteromeric assemblies. This versatility and the general accessibility by de novo design makes them ideal building blocks for synthetic biology. This Minireview highlights the efforts being made in designing small peptide catalysts or reaction templates based on the CC scaffold. The first reports described autocatalysis or mediation of peptide ligation based on CC recognition. Over the years, the designs became more advanced, catalyzing ester hydrolysis, acyl transfer and redox reactions with partial enzyme-like reactivity. Due to the ability to control CC assembly, and, in heterodimeric systems, the association and dissociation, the CC motif has become a common peptide tag in chemical biology.

Decoration of Coiled-Coil Peptides with N-Cysteine Peptide Thioesters As Cyclic Peptide Precursors Using Copper-Catalyzed Azide-Alkyne Cycloaddition (CuAAC) Click Reaction.

The development of a copper-catalyzed azide-alkyne cycloaddition (CuAAC) protocol for the decoration of coiled coils with N-cysteine peptide thioesters as cyclic peptide precursors is presented. The reaction conditions include tert-butanol/PBS as the solvent and CuSO4/THPTA/ascorbate as the catalytic system. During these studies, partial formylation of N-terminal cysteine peptides is observed. Mechanistic analysis leads to identification of the formyl source and, hence, to the development of reaction conditions, under which the undesired side reaction was suppressed.

De Novo-Designed α-Helical Barrels as Receptors for Small Molecules.

We describe de novo-designed α-helical barrels (αHBs) that bind and discriminate between lipophilic biologically active molecules. αHBs have five or more α-helices arranged around central hydrophobic channels the diameters of which scale with oligomer state. We show that pentameric, hexameric, and heptameric αHBs bind the environmentally sensitive dye 1,6-diphenylhexatriene (DPH) in the micromolar range and fluoresce. Displacement of the dye is used to report the binding of nonfluorescent molecules: palmitic acid and retinol bind to all three αHBs with submicromolar inhibitor constants; farnesol binds the hexamer and heptamer; but ß-carotene binds only the heptamer. A co-crystal structure of the hexamer with farnesol reveals oriented binding in the center of the hydrophobic channel. Charged side chains engineered into the lumen of the heptamer facilitate binding of polar ligands: a glutamate variant binds a cationic variant of DPH, and introducing lysine allows binding of the biosynthetically important farnesol diphosphate.