A Minispidroin Guides the Molecular Design for Cellular Condensation Mechanisms in S. cerevisiae.

Structural engineering of molecules for condensation is an emerging technique within synthetic biology. Liquid-liquid phase separation of biomolecules leading to condensation is a central step in the assembly of biological materials into their functional forms. Intracellular condensates can also function within cells in a regulatory manner to facilitate reaction pathways and to compartmentalize interactions. We need to develop a strong understanding of how to design molecules for condensates and how their in vivo-in vitro properties are related. The spider silk protein NT2RepCT undergoes condensation during its fiber-forming process. Using parallel in vivo and in vitro characterization, in this study, we mapped the effects of intracellular conditions for NT2RepCT and its several structural variants. We found that intracellular conditions may suppress to some extent condensation whereas molecular crowding affects both condensate properties and their formation. Intracellular characterization of protein condensation allowed experiments on pH effects and solubilization to be performed within yeast cells. The growth of intracellular NT2RepCT condensates was restricted, and Ostwald ripening was not observed in yeast cells, in contrast to earlier observations in E. coli. Our results lead the way to using intracellular condensation to screen for properties of molecular assembly. For characterizing different structural variants, intracellular functional characterization can eliminate the need for time-consuming batch purification and in vitro condensation. Therefore, we suggest that the in vivo-in vitro understanding will become useful in, e.g., high-throughput screening for molecular functions and in strategies for designing tunable intracellular condensates.

Fortified Coiled Coils: Enhancing Mechanical Stability with Lactam or Metal Staples.

Coiled coils (CCs) are powerful supramolecular building blocks for biomimetic materials, increasingly used for their mechanical properties. Here, we introduce helix-inducing macrocyclic constraints, so-called staples, to tune thermodynamic and mechanical stability of CCs. We show that thermodynamic stabilization of CCs against helix uncoiling primarily depends on the number of staples, whereas staple positioning controls CC mechanical stability. Inserting a covalent lactam staple at one key force application point significantly increases the barrier to force-induced CC dissociation and reduces structural deformity. A reversible His-Ni2+ -His metal staple also increases CC stability, but ruptures upon mechanical loading to allow helix uncoiling. Staple type, position and number are key design parameters in using helical macrocyclic templates for fine-tuning CC properties in emerging biomaterials.

Influence of Network Topology on the Viscoelastic Properties of Dynamically Crosslinked Hydrogels.

Biological materials combine stress relaxation and self-healing with non-linear stress-strain responses. These characteristic features are a direct result of hierarchical self-assembly, which often results in fiber-like architectures. Even though structural knowledge is rapidly increasing, it has remained a challenge to establish relationships between microscopic and macroscopic structure and function. Here, we focus on understanding how network topology determines the viscoelastic properties, i.e., stress relaxation, of biomimetic hydrogels. We have dynamically crosslinked two different synthetic polymers with one and the same crosslink. The first polymer, a polyisocyanopeptide (PIC), self-assembles into semi-flexible, fiber-like bundles, and thus displays stress-stiffening, similar to many biopolymer networks. The second polymer, 4-arm poly(ethylene glycol) (starPEG), serves as a reference network with well-characterized structural and viscoelastic properties. Using one and the same coiled coil crosslink allows us to decouple the effects of crosslink kinetics and network topology on the stress relaxation behavior of the resulting hydrogel networks. We show that the fiber-containing PIC network displays a relaxation time approximately two orders of magnitude slower than the starPEG network. This reveals that crosslink kinetics is not the only determinant for stress relaxation. Instead, we propose that the different network topologies determine the ability of elastically active network chains to relax stress. In the starPEG network, each elastically active chain contains exactly one crosslink. In the absence of entanglements, crosslink dissociation thus relaxes the entire chain. In contrast, each polymer is crosslinked to the fiber bundle in multiple positions in the PIC hydrogel. The dissociation of a single crosslink is thus not sufficient for chain relaxation. This suggests that tuning the number of crosslinks per elastically active chain in combination with crosslink kinetics is a powerful design principle for tuning stress relaxation in polymeric materials. The presence of a higher number of crosslinks per elastically active chain thus yields materials with a slow macroscopic relaxation time but fast dynamics at the microscopic level. Using this principle for the design of synthetic cell culture matrices will yield materials with excellent long-term stability combined with the ability to locally reorganize, thus facilitating cell motility, spreading, and growth.

Where Do Early Career Researchers Stand on Open Science Practices? A Survey Within the Max Planck Society.

Open science (OS) is of paramount importance for the improvement of science worldwide and across research fields. Recent years have witnessed a transition toward open and transparent scientific practices, but there is still a long way to go. Early career researchers (ECRs) are of crucial relevance in the process of steering toward the standardization of OS practices, as they will become the future decision makers of the institutional change that necessarily accompanies this transition. Thus, it is imperative to gain insight into where ECRs stand on OS practices. Under this premise, the Open Science group of the Max Planck PhDnet designed and conducted an online survey to assess the stance toward OS practices of doctoral candidates from the Max Planck Society. As one of the leading scientific institutions for basic research worldwide, the Max Planck Society provides a considerable population of researchers from multiple scientific fields, englobed into three sections: biomedical sciences, chemistry, physics and technology, and human and social sciences. From an approximate total population of 5,100 doctoral candidates affiliated with the Max Planck Society, the survey collected responses from 568 doctoral candidates. The survey assessed self-reported knowledge, attitudes, and implementation of different OS practices, namely, open access publications, open data, preregistrations, registered reports, and replication studies. ECRs seemed to hold a generally positive view toward these different practices and to be interested in learning more about them. Furthermore, we found that ECRs' knowledge and positive attitudes predicted the extent to which they implemented these OS practices, although levels of implementation were rather low in the past. We observed differences and similarities between scientific sections. We discuss these differences in terms of need and feasibility to apply these OS practices in specific scientific fields, but additionally in relation to the incentive systems that shape scientific communities. Lastly, we discuss the implications that these results can have for the training and career advancement of ECRs, and ultimately, for the consolidation of OS practices.

Bioinspired Histidine⁻Zn2+ Coordination for Tuning the Mechanical Properties of Self-Healing Coiled Coil Cross-Linked Hydrogels.

Natural biopolymeric materials often possess properties superior to their individual components. In mussel byssus, reversible histidine (His)-metal coordination is a key feature, which mediates higher-order self-assembly as well as self-healing. The byssus structure, thus, serves as an excellent natural blueprint for the development of self-healing biomimetic materials with reversibly tunable mechanical properties. Inspired by byssal threads, we bioengineered His-metal coordination sites into a heterodimeric coiled coil (CC). These CC-forming peptides serve as a noncovalent cross-link for poly(ethylene glycol)-based hydrogels and participate in the formation of higher-order assemblies via intermolecular His-metal coordination as a second cross-linking mode. Raman and circular dichroism spectroscopy revealed the presence of α-helical, Zn2+ cross-linked aggregates. Using rheology, we demonstrate that the hydrogel is self-healing and that the addition of Zn2+ reversibly switches the hydrogel properties from viscoelastic to elastic. Importantly, using different Zn2+:His ratios allows for tuning the hydrogel relaxation time over nearly three orders of magnitude. This tunability is attributed to the progressive transformation of single CC cross-links into Zn2+ cross-linked aggregates; a process that is fully reversible upon addition of the metal chelator ethylenediaminetetraacetic acid. These findings reveal that His-metal coordination can be used as a versatile cross-linking mechanism for tuning the viscoelastic properties of biomimetic hydrogels.

Tuning coiled coil stability with histidine-metal coordination.

Coiled coils (CCs) have emerged as versatile building blocks for the synthesis of nanostructures, drug delivery systems and biomimetic hydrogels. Bioengineering metal coordination sites into the terminal ends of a synthetic coiled coil (CC), we generate a nanoscale biological building block with tunable stability. The reversible coordination of Ni2+ thermodynamically stabilizes the CC, as shown with circular dichroism spectroscopy. Using atomic force microscopy-based single-molecule force spectroscopy, it is further shown that Ni2+-binding reinforces the CC mechanically, increasing the barrier height for dissociation. When used as a dynamic crosslink in polyethyleneglycol-based hydrogels, the single-molecule stability of the CC is directly transferred to the bulk material and determines its viscoelastic properties. This reversibly tunable CC, thus, highlights an effective strategy for rationally engineering the single-molecule properties of biomolecular building blocks, which can be translated to the emergent properties of biomimetic materials, as well as other CC containing molecular assemblies.

Goodness of fit testing in dynamic single-molecule force spectroscopy.

Dynamic single-molecule force spectroscopy (SMFS) is a powerful method to characterize the mechanical stability of biomolecules. We address the problem that the standard manner of reporting the extracted energy landscape parameters does not reveal the intrinsic statistical errors associated with them. This problem becomes particularly relevant when SMFS is used to compare two or more different molecular systems. Here, we propose two methods that allow for a straightforward test of statistical significance. We illustrate the power of the methods by applying them to the experimental results obtained for three dimeric coiled coils of different lengths. Both methods are general and may be applied to any problem involving the fit of models with two correlated parameters.