Ubiquitin Designer Proteins as a New Additive Generation toward

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Ubiquitin Designer Proteins as a New Additive Generation towards Controlling Crystallization. Cristina Ruiz Agudo, Joachim Lutz, Philipp Keckeis, Michael King, Andreas Marx, and Denis Gebauer J. Am. Chem. Soc., Just Accepted Manuscript • DOI: 10.1021/jacs.9b06473 • Publication Date (Web): 19 Jul 2019 Downloaded from pubs.acs.org on July 19, 2019

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Journal of the American Chemical Society

Ubiquitin Designer Proteins as a New Additive Generation towards Controlling Crystallization. Cristina Ruiz-Agudo†, Joachim Lutz†, Philipp Keckeis†, Michael King†, Andreas Marx†, and Denis Gebauer†#* †Department of Chemistry, University of Konstanz, Konstanz, Germany. #present address: Institute of Inorganic Chemistry, Leibniz University of Hannover, Hannover, Germany

Supporting Information Placeholder ABSTRACT: Proteins controlling mineralization in vivo are diverse, suggesting that there are various ways by which mineralization can be directed in bio-inspired approaches. While well-defined three-dimensional (3D) structures occur in biomineralization proteins, the design of synthetic, soluble, bioinspired macromolecules with specific, reproducible and predictable 3D arrangements of mineral-interacting functions poses an ultimate challenge. Thus, the question how certain arrangements of such functions on protein surfaces influence mineralization, and in which way, subsequently, specific alterations affect this process, remains elusive. Here, we used genetically engineered Ubiquitin (Ub) proteins in order to overcome the limitations of generic bioinspired additive systems. By advancing existing protocols, we introduced an unnatural amino acid and, subsequently, mineralinteracting functions via selective pressure incorporation and click chemistry, respectively, without affecting the Ub secondary structure. Indeed, as-obtained Ub with three phosphate functions at defined positions shows unique effects, based on a yet unmatched capability towards the stabilization of a film of a dense liquid mineral phase visible even by naked eye, its transformation into amorphous nanoparticles, and afterwards crystals with complex shapes. We thereby demonstrate that Ub designer proteins pose a unique, new generation of crystallization additives where the 3D arrangement of mineral-interacting functions can be designed at will, promising a future use for programmable, target-oriented mineralization control.

Sophisticated control over crystallization processes in living organisms is essential for the generation of biominerals —unique organic-inorganic hybrid structures with exceptional mechanical properties.1 Here, proteins can regulate crystal polymorphism, control the location and orientation of biogenic mineral phases, or act as templates providing specific sites for nucleation.2 Previous studies of, e.g., the genome of sea urchins illustrated the variety of biomineralization proteins.3,4 In spicules of S. purpuratus, for instance, the transformation of amorphous calcium carbonate (ACC) into calcite is regulated by a soluble organic matrix composed of more than 45 proteins.5 However, the mechanisms by which crystallization is influenced and controlled in vivo are still under debate,6,7 and have been resolved only for some examples.8– 11

Figure 1. Overview of the main types of crystallization additives. Generic polyelectrolytes exhibit distinct polydispersities and can undergo pHdependent conformational changes (a, top). Altered primary sequences can affect the secondary structure of (a, bottom) peptides, b) intrinsically disordered proteins and c) folded proteins. In the case of Ub designer proteins (d), specific 3D arrangements of mineral-interacting functions are accessible thanks to the stability of the protein conformation. Red circles indicate modifications, whereas only in case of Ub designer proteins, the exact 3D arrangement of mineral-interacting functions (golden spheres in d) can be precisely designed at will. For further explanation, see the text.

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Figure 2. a) Selective pressure incorporation of the unnatural amino acid azidohomoalanine (Aha) in Ubiquitin (Ub) yields UbAha3 (UbN25Aha D32Aha D58Aha) for further specific functionalization. Cu(I)-catalyzed azide alkyne cycloaddition (“Click chemistry”) was employed to modify UbAha3 with the phosphate linker propargyl phosphate resulting in UbP3 (Ub N25Aha(PO4) D32Aha(PO4) D58Aha(PO4)). b) Surface representation of UbP3 with aligned modified positions marked in red. c) CaCO3 crystallization in the presence of UbP3 was investigated via a droplet-based gas diffusion method. The crystallization process was significantly influenced by the presence of the functionalized UbP3 protein. A macroscopic CaCO3 liquid phase was stabilized for some hours and subsequently consumed yielding amorphous calcium carbonate nanoparticles (ACC Np). Due to the vapor diffusion method, the calcium carbonate concentration prior phase separation is highest near the droplet surface, where the precursor film forms initially. Subsequently forming particles thus likely emerge at or near the firm too, but might later sediment into the bulk of the droplets as shown.

systems has been extensively investigated (e.g. Au, CdS, CdSe, ZnS, ZnSe, Fe3O4).31–34 In the work presented here, Ubiquitin (Ub) designer proteins (Figure 1d) were modified in a spacial-controlled manner with functionalities that are known to interact with CaCO3 crystallization precursors and intermediates, directing crystallization. The high structural stability and ruggedness against modifications renders Ub an optimal candidate for this challenging endeavor. Ub is a globular 76 amino acids protein with a melting point near 100 °C at neutral pH.35 In this pioneering study, we selected phosphate functionalization to be introduced into Ub (Figure 2a and Figure S1) since 36 generic phosphate-based additives also exert strong effects on calcium carbonate crystallization.18,37 Moreover, phosphorylation is one of the most widespread post-translational modification of proteins occurring in the organic matrix of biominerals.38–41 Phosphorylated proteins are suggested to be relevant in directing calcium carbonate42,43 and phosphate44 mineralization due to the great affinity of phosphate for Ca2+ ions45 that can stabilize solid46 and liquid precursor phases.44 In order to design the Ub protein, first, we inserted three azido functionalities using the unnatural amino acid azidohomoalanine (Aha) and selective pressure incorporation by refining established protocols (Figure 2a and Figure S2).47–49 Subsequently, the azide functionalities were modified with phosphates by Cu-catalyzed alkyne-azide cycloaddition (“Click Chemistry”). This results in a well-defined linear arrangement of the mineral interacting phosphate functions on one side of the macromolecule (Figure 2b). Ub N25Aha D32Aha D58Aha (Figure S3, UbAha3 where Aha=azidohomoalanine), served as a reference, as it does not contain the phosphate-functionalization of the Ub analogue that was at the heart of this study, UbN25Aha+PO4 D32Aha+PO4 D58Aha+PO4 (UbP3). Having these compounds in hands, the effects of the Ub variants during crystallization were investigated by a droplet-based gas diffusion method inspired by previous studies (Figure 2c).50 The specific introduction of phosphate groups on UbP3 resulted in the

Synthetic additives with functional groups and motifs typically found in biomineralization proteins are able to delicately influence in vitro crystallization2,12–14 where the most abundant biomineral, CaCO3, is also of vast industrial and geological importance.15 Bioinspired additives used in CaCO3 crystallization range from simple ions or molecules (e.g. Mg2+ 16, citrate17, phosphate18, amino acids19) over macromolecules such as polymers (mainly polyelectrolytes)20 and peptides,21 intrinsically disordered proteins,22 to folded proteins23,24 (Figure 1). Changes in the environment, such as temperature and pH (Figure 1a), or binding events and interactions with mineral surfaces, can alter the conformation of common polymeric additives (Figure 1a) in an unpredictable manner.25 These additives, except peptides and proteins, also possess no specifically tailored —or tailorable— primary or secondary structures, and exhibit significant polydispersity.21 Folded, monodisperse proteins play crucial roles in biomineralization,23,26,27 highlighting that the 3D arrangement of mineral-interacting functions in structurally well-defined species contributes to achieving sophisticated mineralization controls. How certain arrangements of such functions influence mineralization, and in which way specific alterations affect this process, however, remains elusive. This is due to the limitations of generic additive systems: No specific alterations of any identified 3D arrangements of mineral-interacting functions on protein surfaces can be designed at will because changes in primary sequences (indicated by red circles in Figure 1) affect the secondary structures (Figure 1a-c). While effects of designer peptides21,28,29 and modified proteins30 have been previously studied, no allusion to the potential influence of the macromolecules during the early stages of crystallization of CaCO3 was made, whereas any subsequent, specific changes of the 3D arrangements of mineralinteractions were not attempted, owing to the inherent restrictions outlined above. Here, we design a new generation additive system in order to overcome the inherent limitations of the previous ones. The ability of engineered proteins and viruses to interact with diverse inorganic

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Journal of the American Chemical Society unique formation of a macroscopic CaCO3 film visible even by naked eye (at UbP3 concentrations above 0.1 mg mL-1), while there were only minor effects at 0.01 mg mL-1 (Figure S4). The approximately 100 nm thick film (Figure 3) composed of C, O and Ca (Figure S5), exhibited no evidence of crystallinity in selected area electron diffraction (SAED, inset in Figure 3c) or ATR-IR spectra (Figure S6 and S7). On the other hand, in the reference experiments, calcite crystals and intermediates reminiscent of precursors to calcite crystals with platonic shapes51 coexisted with a few vaterite particles (Figure S8, S9, S10). Very similar CaCO3 crystals as found in the UbAha3 reference samples had already been reported in the presence of engineered peptides21,28,30, nacre proteins52 and block co-polymers.51 In contrast, UbP3 strongly stabilizes a macroscopic calcium carbonate film (Figure S11) considerably larger than previously obtained CaCO3 films in the presence of polymeric additives12,13,53 and biomineralization proteins.54 In order to shed more light on the film formation and structure in presence of UbP3, the gas diffusion experiment was emulated within an Environmental Scanning Electron Microscope (ESEM) chamber (details in Supporting information, Figure S12). In the controls (Figure S13a and b), the results resembled those of the regular gas diffusion experiments without proteins (see below). With 1 mg mL-1 UbP3, an undulating structure occurred after ca. 30 min (Figure 3d), resembling waves also seen by naked eye during regular gas diffusion experiments (Figure S14). Afterwards, rounded shapes occurred underneath or within this film (Figure S13c), which eventually partly disappeared as CaCO3 fractal structures emerged (Figure S13d). In samples that were isolated from regular droplet-based gas diffusion experiments the initially formed film also disappeared after a few hours, resulting in the formation of amorphous CaCO3 (ACC), vaterite and calcite (Figure S15). Irradiation of isolated films under the TEM beam triggered crystallization of calcium carbonate, confirming that it was indeed a CaCO3 precursor phase (Figure S16). In CO2-free environments, no film developed upon NH3 diffusion into a drop containing CaCl2 and UbP3 (Figure S17), revealing that it is not formed by UbP3 and calcium ions upon an increase in pH, which may then serve as a substrate for heterogeneous nucleation of ACC. The macroscopic precursor CaCO3 film (Figure 4a) transformed into ca. 100-200 nm sized ACC (Figure S18) nanoparticles probably upon the loss of significant amounts of water (Figure 4b). In some locations, the ACC nanoparticles evolved to form larger entities (Figure 4c), which eventually transformed into vaterite particles (Figure 4d). Approximately two hours after its detection, the film was totally consumed due to the formation of ACC nanoparticles (Figure 4e) and vaterite (Figure 4f). This implies that the film is constituted by a CaCO3 liquid condensed phase (LCP) that was temporally stabilized by UbP3, commonly known as polymer-induced liquid precursors (PILPs,12 which are additive-stabilized rather thaninduced states55–57). There is evidence for the liquid character of the film that is rather compelling: A macroscopic phase of at least 100 nm thickness transforms into smaller nanoparticles, which is thermodynamically impossible if the film was solid ACC. A liquid film, by contrast, changes its composition according to that of the mother liquid, until it is consumed upon particle formation when the liquid-liquid binodal limit57 is eventually undercut —as observed. Alternatively, the film might be built up of very small solid nanoparticles, cross-bridged by proteins. However, in this case, it should be straightforward to isolate the film —which was impossible to achieve in multiple attempts using tweezers, glass slides, or blades. The film dissolved upon mechanical manipulation towards isolation, as a dense liquid can only exist in equilibrium with the mother liquid.

Figure 3. CaCO3 film formed in the presence of UbP3. a) and b) SEM images of the film. c) TEM image of the film and ACC nanoparticles; SAED (inset) confirms the amorphous character of the film. d) In-situ STEM image of the formation of the film inside the ESEM chamber after 30 min.

The fact that no film was observed in the reference experiments, while it formed at all UbP3 concentrations above ca. 0.1 mg mL-1, implies that specific interactions introduced between UbP3 and the CaCO3 LCP facilitate the formation of a spatially extended PILP at the air-liquid interface. Since the CaCO3 film was also not formed when an equivalent concentration of the linker molecule carrying phosphate groups was present in the solution (Figure S19), it is also obvious that their specific geometric arrangement, resulting in a close proximity of phosphate groups on the UbP3 surface, is important. However, for generic additives that are potent PILP stabilizers, the formation of macroscopic films has not been reported previously, to the best of our knowledge.2 The functionalization of Ub with PO43- groups (UbP3) introduces negative charges which allow strong interactions with calcium ions as well as forming calcium carbonate phases. It was shown that upon entering the liquid-liquid miscibility gap, pre-nucleation clusters become LCP-nanodroplets larger than ca. 2 nm in size, which subsequently aggregate to form growing LCP intermediates that subsequently dehydrate yielding amorphous nanoparticles.57,58 We propose that due to the similar size of UbP3 and initially formed nanodroplets, and the strong interactions of the phosphate groups of UbP3 with the calcium carbonate phase, the protein is regularly integrated into the LCP growing via aggregation, thereby effectively stabilizing it towards the generation of a macroscopic film. This implies that the protein is incorporated into ACC particles, and later, crystals, too. After 5h to 24 h a mixture of calcite and vaterite (Figure S15) was obtained from the film and ACC precursors, displaying exceptional and complex features (Figure S20a and b). Bundles of rods are densely arranged and partially fused on the spherical vaterite particles’ surface (Figure S20a and c). Likewise, prismatic submicrometer units and rod bundles build up the rhombohedral CaCO3 particles (Figure S20b and d). While also the reference samples stabilize nanoparticle subunits (Figure S10), this effect is significantly more pronounced for UbP3, probably due to the strong binding of mineral precursors and intermediates by the phosphate groups.



Notes The authors declare no competing financial interests.

ACKNOWLEDGMENT This work was performed within the framework of the SFB 1214 (project A07) funded by the German Research Foundation (DFG). DG was a Research Fellow of the Zukunftskolleg of the University of Konstanz during this work. CRA thanks the SFB 1214 and the Zukunftskolleg of the University of Konstanz for financial support. We would like to thank the personnel of the “Centro de Instrumentación Científica” (University of Granada) for their support and help with the ESEM, FESEM and TEM analyses. In addition, we would like to thank the Particle Analysis Center of the University of Konstanz (funded by SFB1214). We also thank Prof. Dr. Christine Peter for comments that greatly improved the manuscript.

REFERENCES (1) Lowenstam, H. A.; Weiner, S. On biomineralization; Oxford University Press: New York, 1989. (2) L. B. Gower. Biomimetic Model Systems for Investigating the Amorphous Precursor Pathway and Its Role in Biomineralization. Chem. Rev. 2008, 108, 4551–4627. (3) Sodergren, E.; Weinstock, G. M.; Davidson, E. H.; Cameron, R. A.; Gibbs, R. A.; Angerer, R. C. et al. The Genome of the Sea Urchin Strongylocentrotus purpuratus. Science 2006, 314, 941– 952. (4) Zhang, G.; Fang, X.; Guo, X.; Li, L.; Luo, R.; Xu, F.; et al. The Oystergenome Reveals Stress Adaptation and Complexity of Shell Formation. Nature 2012, 490, 49–54. (5) Raz, S.; Hamilton, P. C.; Wilt, F. H.; Weiner, S.; Addadi, L. The Transient Phase of Amorphous Calcium Carbonate in Sea Urchin Larval Spicules: The Involvement of Proteins and Magnesium Ions in Its Formation and Stabilization. Adv. Funct. Mater. 2003, 13, 480–486. (6) Evans, J. S. "Liquid-Like" Biomineralization Protein Assemblies: A Key to the Regulation of Non-Classical Nucleation. CrystEngComm 2013, 15, 8388–8394. (7) Nudelman, F.; Sommerdijk, N. Biomineralization as an Inspiration for Materials Chemistry. Angew. Chem. Int. Ed. 2012, 51, 6582–6596. (8) Politi, Y.; Arad, T.; Klein, E.; Weiner, S.; Addadi, L. Sea Urchin Spine Calcite Forms via a Transient Amorphous Calcium Carbonate Phase. Science 2004, 306, 1161–1164. (9) Bahn, S. Y.; Jo, B. H.; Choi, Y. S.; Cha, H. J. Control of Nacre Biomineralization by Pif80 in Pearl Oyster. Sci. Adv. 2017, 3, e1700765. (10) Seto, J.; Ma, Y.; Davis, S. A.; Meldrum, F.; Gourrier, A.; Kim, Y.-Y.; Schilde, U.; Sztucki, M.; Burghammer, M.; Maltsev, S. et al. Structure-Property Relationships of a Biological Mesocrystal in the Adult Sea Urchin Spine. PNAS 2012, 109, 3699–3704. (11) Belcher, A. M.; Wu, X. H.; Christensen, R. J.; Hansma, P. K.; Stucky, G. D.; Morse, D. E. Control of Crystal Phase Switching and Orientation by Soluble Mollusc-Shell Proteins. Nature 1996, 381, 56–58. (12) Gower, L. B.; Odom, D. J. Deposition of Calcium Carbonate Films by a Polymer-Induced Liquid-Precursor (PILP) Process. J. Cryst. Growth 2000, 210, 719–734. (13) Gower, L. A.; Tirrell, D. A. Calcium Carbonate Films and Helices Grown in Solutions of Poly(Aspartate). J. Cryst. Growth 1998, 191, 153–160. (14) Meldrum, F. C.; Cölfen, H. Controlling Mineral Morphologies and Structures in Biological and Synthetic Systems. Chem. Rev. 2008, 108, 4332–4432.

Figure 4. Evolution of the CaCO3 film upon water loss. a) Thin film and scarce ACC nanoparticles. b) The film became thicker and 100-200 nm spheres formed (inset); c) Formation of larger rounded entities in some parts of the film. d) Partial consumption of the film after 2h yielded e) 100-200 nm CaCO3 spheres and f) larger rounded particles (approx. 20-µm diameter).

Our results demonstrate that engineered Ub proteins can strongly influence CaCO3 crystallization by stabilizing liquid precursor phases. The introduction of three phosphate groups yielding UbP3 led to an unprecedented capability towards LCP stabilization yielding a macroscopic PILP film even visible by naked eye. This and the subsequent formation of crystals with complex shapes from the precursor film highlights the potential use of designer proteins in crystallization control. Future studies will reveal if altered positions and chemistries of the Ub functionalization will cause distinct effects, and whether these can be rationalized based upon corresponding changes in both three-dimensional arrangement and directionality of organic-inorganic interactions. In any case, we demonstrate that genetically-engineered Ub designer proteins are a new generation of additives that may facilitate target-oriented and programmable control of crystallization processes in the future.

ASSOCIATED CONTENT Supporting Information Detailed description of experimental procedures and additional analytical data. The Supporting Information is available free of charge on the ACS Publications website.

AUTHOR INFORMATION Corresponding Author [email protected]


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Journal of the American Chemical Society (15) Geyssant, J.; Huwald, E.; Strauch, D. Calcium carbonate: from the Cretaceous period into the 21st century; Tegethoff, F. W., Ed.; Birkhäuser Verlag: Basel, Boston, Berlin, 2001. (16) Politi, Y.; Batchelor, D. R.; Zaslansky, P.; Chmelka, B. F.; Weaver, J. C.; Sagi, I.; Weiner, S. Addadi, L. Role of Magnesium Ion in the Stabilization of Biogenic Amorphous Calcium Carbonate: A Structure−Function Investigation. Chem. Mater. 2010, 22, 161–166. (17) Tobler, D. J.; Rodriguez-Blanco, J. D.; Dideriksen, K.; Bovet, N.; Sand, K. K.; Stipp, S. L. S. Citrate Effects on Amorphous Calcium Carbonate (ACC) Structure, Stability, and Crystallization. Adv. Funct. Mater. 2015, 25, 3081–3090. (18) Kababya, S.; Gal, A.; Kahil, K.; Weiner, S.; Addadi, L.; Schmidt, A. Phosphate–Water Interplay Tunes Amorphous Calcium Carbonate Metastability: Spontaneous Phase Separation and Crystallization vs Stabilization Viewed by Solid State NMR. J. Am. Chem. Soc. 2015, 137, 990–998. (19) Tobler, D. J.; Blanco, J. R.; Dideriksen, K.; Sand, K. K.; Bovet, N.; Benning, L. G.; Stipp, S.L.S. The Effect of Aspartic Acid and Glycine on Amorphous Calcium Carbonate (ACC) Structure, Stability and Crystallization. Procedia Earth Planet. Sci. 2014, 10, 143–148. (20) Yu, S.-H.; Cölfen, H. Bio-inspired Crystal Morphogenesis by Hydrophilic Polymers. J. Mater. Chem. 2004, 14, 2124–2147. (21) Chen, C.-L.; Qi, J.; Zuckermann, R. N.; DeYoreo, J. J. Engineered Biomimetic Polymers as Tunable Agents for Controlling CaCO3 Mineralization. J. Am. Chem. Soc. 2011, 133, 5214–5217. (22) Rao, A.; Drechsler, M.; Schiller, S.; Scheffner, M.; Gebauer, D.; Cölfen, H. Stabilization of Mineral Precursors by Intrinsically Disordered Proteins. Adv. Funct. Mater. 2018, 28, 1802063. (23) Ruiz-Arellano, R. R.; Medrano, F. J.; Moreno, A.; Romero, A. Structure of Struthiocalcin-1, an Intramineral Protein from Struthio Camelus Eggshell, in Two Crystal Forms. Acta. Crystallogr. D 2015, 71, 809–818. (24) Pipich, V.; Balz, M.; Wolf, S. E.; Tremel, W.; Schwahn, D. Nucleation and Growth of CaCO3 Mediated by the Egg-White Protein Ovalbumin: A Time-Resolved in situ Study Using SmallAngle Neutron Scattering. J. Am. Chem. Soc. 2008, 130, 6879– 6892. (25) Grant, B. J.; Gorfe, A.; McCammon, J. A. Large Conformational Changes in Proteins: Signaling and Other Functions. Curr. Opin. Struct. Biol. 2010, 20, 142–147. (26) Colin L. Freeman; John H. Harding; David Quigley; P. Mark Rodger. Structural Control of Crystal Nuclei by an Eggshell Protein. Angew. Chem. Int. Ed. 2010, 49, 5135–5137. (27) Lakshminarayanan, R.; Chi-Jin, E. O.; Loh, X. J.; Kini, R. M.; Valiyaveettil, S. Purification and Characterization of a Vaterite-Inducing Peptide, Pelovaterin, from the Eggshells of Pelodiscus sinensis (Chinese Soft-Shelled Turtle). Biomacromolecules 2005, 6, 1429–1437. (28) DeOliveira, D. B.; Laursen, R. A. Control of Calcite Crystal Morphology by a Peptide Designed To Bind to a Specific Surface. J. Am. Chem. Soc. 1997, 119, 10627–10631. (29) Masica, D. L.; Schrier, S. B.; Specht, E. A.; Gray, J. J. De Novo Design of Peptide−Calcite Biomineralization Systems. J. Am. Chem. Soc. 2010, 132, 12252–12262. (30) Heinonen, H.; Laaksonen, P.; Markus, B. Linder, M. B.; Hentze, H.-P. Engineered Hydrophobin for Biomimetic Mineralization of Functional Calcium Carbonate Microparticles. J. Biomater. Nanobiotechnol. 2014, 5, 1–7. (31) Uchida, M.; Flenniken, M. L.; Allen, M.; Willits, D. A.; Crowley, B. E.; Brumfield, S.; Willis, A. F.; Jackiw, L.; Jutila, M.; Young, M. J. et al. Targeting of Cancer Cells with Ferrimagnetic Ferritin Cage Nanoparticles. J. Am. Chem. Soc. 2006, 128, 16626–16633.

(32) Douglas, T.; Dickson, D. P. E.; Betteridge, S.; Charnock, J.; Garner, C. D.; Mann, S. Synthesis and Structure of an Iron(III) Sulfide-Ferritin Bioinorganic Nanocomposite. Science 1995, 269, 54–57. (33) Whaley, S. R.; English, D. S.; Hu, E. L.; Barbara, P. F.; Belcher, A. M. Selection of Peptides with Semiconductor Binding Specificity for Directed Nanocrystal Assembly. Nature 2000, 405, 665-668. (34) Peelle, B. R.; Krauland, E. M.; Wittrup, K. D.; Belcher, A. M. Design Criteria for Engineering Inorganic Material- Specific Peptides. Langmuir 2005, 21, 6929–6933. (35) Crespo, M. D.; Rubini, M. Rational Design of Protein Stability: Effect of (2S,4R)-4-Fluoroproline on the Stability and Folding Pathway of Ubiquitin. PloS one 2011, 6, e19425. (36) Vinogradov, A. P. Chemical Composition of Marine Organisms; CT: New Haven, 1953. (37) Gal, A.; Kahil K.; Vidavsky N.; DeVol R. T.; Gilbert, P. P. A.U.; Fratzl P.; Weiner, S.; Addadi, L. Particle Accretion Mechanism Underlies Biological Crystal Growth from an Amorphous Precursor Phase. Adv. Funct. Mater. 2014, 24, 5420– 5426. (38) Tagliabracci, V. S.; Engel, J. L.; Wen, J.; Wiley, S. E.; Worby, C. A.; Kinch, L. N.; Xiao, J.; Grishin, N. V.; Dixon, J. E. Secreted Kinase Phosphorylates Extracellular Proteins That Regulate Biomineralization. Science 2012, 336, 1150–1153. (39) Gericke, A.; Qin, C.; Spevak, L.; Fujimoto, Y.; Butler, W. T.; Sørensen, E. S.; Boskey, A. L. Importance of Phosphorylation for Osteopontin Regulation of Biomineralization. Calcif. Tissue. Int. 2005, 77, 45–54. (40) Mann, K.; Olsen, J. V.; Maček, B.; Gnad, F.; Mann, M. Phosphoproteins of the Chicken Eggshell Calcified Layer. Proteomics 2007, 7, 106–115. (41) Narayanan, K.; Ramachandran, A.; Hao, J.; He, G.; Park, K. W.; Cho, M.; George, A. Dual Functional Roles of Dentin Matrix Protein 1: Implications in Biomineralization and Gene Transcription by Activation of Intracellular Ca2+ Store. J. Biol. Chem. 2003, 278, 17500–17508. (42) Wojtas, M.; Wołcyrz, M.; Ożyhar, A.; Dobryszycki, P. Phosphorylation of Intrinsically Disordered Starmaker Protein Increases Its Ability to Control the Formation of Calcium Carbonate Crystals. Cryst. Growth. Des. 2012, 12, 158–168. (43) Mann, K.; Siedler, F. Amino Acid Sequences and Phosphorylation Sites of Emu and Rhea Eggshell C-Type LectinLike Proteins. Comp. Biochem. Physiol. B Biochem. Mol. Biol. 2006, 143, 160–170. (44) Ibsen, C. J. S.; Gebauer, D.; Birkedal, H. Osteopontin Stabilizes Metastable States Prior to Nucleation during Apatite Formation. Chem. Mater. 2016, 28, 8550–8555. (45) Hecker, A.; Testenière, O.; Marin, F.; Luquet, G. Phosphorylation of Serine Residues Is Fundamental for the Calcium-Binding Ability of Orchestin, a Soluble Matrix Protein from Crustacean Calcium Storage Structures. FEBS Lett. 2003, 535, 49–54. (46) Bentov, S.; Weil, S.; Glazer, L.; Sagi, A.; Berman, A. Stabilization of Amorphous Calcium Carbonate by Phosphate Rich Organic Matrix Proteins and by Single Phosphoamino Acids. J. Struct. Bio. 2010, 171, 207–215. (47) Schneider, T.; Schneider, D.; Rösner, D.; Malhotra, S.; Mortensen, F.; Mayer, T. U.; Scheffner, M.; Marx, A. Dissecting Ubiquitin Signaling with Linkage-Defined and Protease Resistant Ubiquitin Chains. Angew. Chem. Int. Ed. 2014, 53, 12925–12929. (48) Rösner, D.; Schneider, T.; Schneider, D.; Scheffner, M.; Marx, A. Click Chemistry for Targeted Protein Ubiquitylation and Ubiquitin Chain Formation. Nat. Protoc. 2015, 10, 1594–1611. (49) Eger, S.; Scheffner, M.; Marx, A.; Rubini, M. Synthesis of Defined Ubiquitin Dimers. J. Am. Chem. Soc. 2010, 132, 16337– 16339.



(50) Ihli, J.; Bots, P.; Kulak, A.; Benning, L. G.; Meldrum, F. C. Elucidating Mechanisms of Diffusion-Based Calcium Carbonate Synthesis Leads to Controlled Mesocrystal Formation. Adv. Funct. Mater. 2013, 23, 1965–1973. (51) Song, R.‐Q.; Xu, A.‐W.; Antonietti, M.; Cölfen, H. Calcite Crystals with Platonic Shapes and Minimal Surfaces. Angew. Chem. Int. Ed. 2009, 48, 395–399. (52) Fu, G.; Valiyaveettil, S.; Wopenka, B.; Morse, D. E. CaCO3 Biomineralization: Acidic 8-kDa Proteins Isolated from Aragonitic Abalone Shell Nacre Can Specifically Modify Calcite Crystal Morphology. Biomacromolecules 2005, 6, 1289–1298. (53) Harris, J.; Mey, I.; Hajir, M.; Mondeshki, M.; Wolf, S. E. Pseudomorphic Transformation of Amorphous Calcium Carbonate Films Follows Spherulitic Growth Mechanisms and Can Give Rise to Crystal Lattice Tilting. CrystEngComm 2015, 17, 6831–6837. (54) Wolf, S. E.; Lieberwirth, I.; Natalio, F.; Bardeau, J.-F.; Delorme, N.; Emmerling, F.; Barrea, R.; Kappl, M.; Marin, F. Merging Models of Biomineralisation with Concepts of Nonclassical Crystallisation: Is a Liquid Amorphous Precursor Involved in The Formation of the Prismatic Layer of the

Mediterranean Fan Mussel Pinna nobilis? Faraday Discuss. 2012, 159, 433–448. (55) Bewernitz, M. A.; Gebauer, D.; Long, J.; Cölfen, H.; Gower, L. B. A Metastable Liquid Precursor Phase of Calcium Carbonate and Its Interactions with Polyaspartate. Faraday Discuss. 2012, 159, 291–312. (56) Wolf, S. E.; Muller, L.; Barrea, R.; Kampf, C. J.; Leiterer, J.; Panne, U.; Hoffmann, T.; Emmerling, F.; Tremel, W. Carbonate-Coordinated Metal Complexes Precede the Formation of Liquid Amorphous Mineral Emulsions of Divalent Metal Carbonates. Nanoscale 2011, 3, 1158–1165. (57) Sebastiani, F.; Wolf, S. L. P.; Born, B.; Luong, T. Q.; Cölfen, H.; Gebauer, D.; Havenith, M. Water Dynamics from THz Spectroscopy Reveal the Locus of a Liquid–Liquid Binodal Limit in Aqueous CaCO3 Solutions. Angew. Chem. Int. Ed. 2017, 56, 490–495. (58) Gebauer, D.; Kellermeier, M.; Gale, J. D.; Bergstrom, L.; Cölfen, H. Pre-nucleation Clusters as Solute Precursors in Crystallisation. Chem. Soc. Rev. 2014, 43, 2348–2371.

TOC Genetically engineered ubiquitin (Ub) proteins strongly impact CaCO3 crystallization. Conjugations of phosphate functions at defined positions on the Ub surface leads to the stabilization of a calcium carbonate dense liquid phase (CaCO3 film) to an extent that is unprecedented. The film transforms into amorphous CaCO3 nanoparticles and subsequently crystals with complex morphologies. Ub designer proteins are presented here as a new generation of additives that may be used as programmable, target-oriented crystallization modifiers in the future: Additive-controlled crystallization 4.0.


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Ubiquitin Designer Proteins as a New Additive Generation toward

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