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- ItemControlling Optical and Catalytic Activity of Genetically Engineered Proteins by Ultrasound(Weinheim : Wiley-VCH, 2021) Zhou, Yu; Huo, Shuaidong; Loznik, Mark; Göstl, Robert; Boersma, Arnold J.; Herrmann, AndreasUltrasound (US) produces cavitation-induced mechanical forces stretching and breaking polymer chains in solution. This type of polymer mechanochemistry is widely used for synthetic polymers, but not biomacromolecules, even though US is biocompatible and commonly used for medical therapy as well as in vivo imaging. The ability to control protein activity by US would thus be a major stepping-stone for these disciplines. Here, we provide the first examples of selective protein activation and deactivation by means of US. Using GFP as a model system, we engineer US sensitivity into proteins by design. The incorporation of long and highly charged domains enables the efficient transfer of force to the protein structure. We then use this principle to activate the catalytic activity of trypsin by inducing the release of its inhibitor. We expect that this concept to switch “on” and “off” protein activity by US will serve as a blueprint to remotely control other bioactive molecules. © 2020 The Authors. Angewandte Chemie International Edition published by Wiley-VCH GmbH
- ItemActivation of the Catalytic Activity of Thrombin for Fibrin Formation by Ultrasound(Weinheim : Wiley-VCH, 2021) Zhao, Pengkun; Huo, Shuaidong; Fan, Jilin; Chen, Junlin; Kiessling, Fabian; Boersma, Arnold J.; Göstl, Robert; Herrmann, AndreasThe regulation of enzyme activity is a method to control biological function. We report two systems enabling the ultrasound-induced activation of thrombin, which is vital for secondary hemostasis. First, we designed polyaptamers, which can specifically bind to thrombin, inhibiting its catalytic activity. With ultrasound generating inertial cavitation and therapeutic medical focused ultrasound, the interactions between polyaptamer and enzyme are cleaved, restoring the activity to catalyze the conversion of fibrinogen into fibrin. Second, we used split aptamers conjugated to the surface of gold nanoparticles (AuNPs). In the presence of thrombin, these assemble into an aptamer tertiary structure, induce AuNP aggregation, and deactivate the enzyme. By ultrasonication, the AuNP aggregates reversibly disassemble releasing and activating the enzyme. We envision that this approach will be a blueprint to control the function of other proteins by mechanical stimuli in the sonogenetics field. © 2021 The Authors. Angewandte Chemie International Edition published by Wiley-VCH GmbH
- ItemReversibly Photo-Modulating Mechanical Stiffness and Toughness of Bioengineered Protein Fibers(Weinheim : Wiley-VCH, 2020) Sun, Jing; Ma, Chao; Maity, Sourav; Wang, Fan; Zhou, Yu; Portale, Giuseppe; Göstl, Robert; Roos, Wouter H.; Zhang, Hongjie; Liu, Kai; Herrmann, AndreasLight-responsive materials have been extensively studied due to the attractive possibility of manipulating their properties with high spatiotemporal control in a non-invasive fashion. This stimulated the development of a series of photo-deformable smart devices. However, it remained a challenge to reversibly modulate the stiffness and toughness of bulk materials. Here, we present bioengineered protein fibers and their optomechanical manipulation by employing electrostatic interactions between supercharged polypeptides (SUPs) and an azobenzene (Azo)-based surfactant. Photo-isomerization of the Azo moiety from the E- to Z-form reversibly triggered the modulation of tensile strength, stiffness, and toughness of the bulk protein fiber. Specifically, the photo-induced rearrangement into the Z-form of Azo possibly strengthened cation–π interactions within the fiber material, resulting in an around twofold increase in the fiber's mechanical performance. The outstanding mechanical and responsive properties open a path towards the development of SUP-Azo fibers as smart stimuli-responsive mechano-biomaterials. © 2020 The Authors. Angewandte Chemie International Edition published by Wiley-VCH GmbH
- ItemModular and Versatile Trans-Encoded Genetic Switches(Weinheim : Wiley-VCH, 2020) Paul, Avishek; Warszawik, Eliza M.; Loznik, Mark; Boersma, Arnold J.; Herrmann, AndreasCurrent bacterial RNA switches suffer from lack of versatile inputs and are difficult to engineer. We present versatile and modular RNA switches that are trans-encoded and based on tRNA-mimicking structures (TMSs). These switches provide a high degree of freedom for reengineering and can thus be designed to accept a wide range of inputs, including RNA, small molecules, and proteins. This powerful approach enables control of the translation of protein expression from plasmid and genome DNA. © 2020 The Authors. Published by Wiley-VCH Verlag GmbH & Co. KGaA
- ItemFour-Dimensional Deoxyribonucleic Acid–Gold Nanoparticle Assemblies(Weinheim : Wiley-VCH, 2020) Luo, Ming; Xuan, Mingjun; Huo, Shuaidong; Fan, Jilin; Chakraborty, Gurudas; Wang, Yixi; Zhao, Hui; Herrmann, Andreas; Zheng, LifeiOrganization of gold nanoobjects by oligonucleotides has resulted in many three-dimensional colloidal assemblies with diverse size, shape, and complexity; nonetheless, autonomous and temporal control during formation remains challenging. In contrast, living systems temporally and spatially self-regulate formation of functional structures by internally orchestrating assembly and disassembly kinetics of dissipative biomacromolecular networks. We present a novel approach for fabricating four-dimensional gold nanostructures by adding an additional dimension: time. The dissipative character of our system is achieved using exonuclease III digestion of deoxyribonucleic acid (DNA) fuel as an energy-dissipating pathway. Temporal control over amorphous clusters composed of spherical gold nanoparticles (AuNPs) and well-defined core–satellite structures from gold nanorods (AuNRs) and AuNPs is demonstrated. Furthermore, the high specificity of DNA hybridization allowed us to demonstrate selective activation of the evolution of multiple architectures of higher complexity in a single mixture containing small and larger spherical AuNPs and AuNRs. © 2020 The Authors. Published by Wiley-VCH Verlag GmbH & Co. KGaA
- ItemCharacterization of Fluorescent Proteins with Intramolecular Photostabilization*(Weinheim : Wiley-VCH, 2021) Henrikus, Sarah S.; Tassis, Konstantinos; Zhang, Lei; van der Velde, Jasper H. M.; Gebhardt, Christian; Herrmann, Andreas; Jung, Gregor; Cordes, ThorbenGenetically encodable fluorescent proteins have revolutionized biological imaging in vivo and in vitro. Despite their importance, their photophysical properties, i. e., brightness, count-rate and photostability, are relatively poor compared to synthetic organic fluorophores or quantum dots. Intramolecular photostabilizers were recently rediscovered as an effective approach to improve photophysical properties of organic fluorophores. Here, direct conjugation of triplet-state quenchers or redox-active substances creates high local concentrations of photostabilizer around the fluorophore. In this paper, we screen for effects of covalently linked photostabilizers on fluorescent proteins. We produced a double cysteine mutant (A206C/L221C) of α-GFP for attachment of photostabilizer-maleimides on the β-barrel near the chromophore. Whereas labelling with photostabilizers such as trolox, a nitrophenyl group, and cyclooctatetraene, which are often used for organic fluorophores, had no effect on α-GFP-photostability, a substantial increase of photostability was found upon conjugation to azobenzene. Although the mechanism of the photostabilizing effects remains to be elucidated, we speculate that the higher triplet-energy of azobenzene might be crucial for triplet-quenching of fluorophores in the blue spectral range. Our study paves the way for the development of fluorescent proteins with photostabilizers in the protein barrel by methods such as unnatural amino acid incorporation. © 2021 The Authors. ChemBioChem published by Wiley-VCH GmbH
- ItemSupercharged Proteins and Polypeptides(Weinheim : Wiley-VCH, 2020) Ma, Chao; Malessa, Anke; Boersma, Arnold J.; Liu, Kai; Herrmann, AndreasElectrostatic interactions play a vital role in nature. Biomacromolecules such as proteins are orchestrated by electrostatics, among other intermolecular forces, to assemble and organize biochemistry. Natural proteins with a high net charge exist in a folded state or are unstructured and can be an inspiration for scientists to artificially supercharge other protein entities. Recent findings show that supercharging proteins allows for control of their properties such as temperature resistance and catalytic activity. One elegant method to transfer the favorable properties of supercharged proteins to other proteins is the fabrication of fusions. Genetically engineered, supercharged unstructured polypeptides (SUPs) are just one promising fusion tool. SUPs can also be complexed with artificial entities to yield thermotropic and lyotropic liquid crystals and liquids. These architectures represent novel bulk materials that are sensitive to external stimuli. Interestingly, SUPs undergo fluid–fluid phase separation to form coacervates. These coacervates can even be directly generated in living cells or can be combined with dissipative fiber assemblies that induce life-like features. Supercharged proteins and SUPs are developed into exciting classes of materials. Their synthesis, structures, and properties are summarized. Moreover, potential applications are highlighted and challenges are discussed. © 2020 The Authors. Published by WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim