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Abstract: Keratin is an abundant structural fibrous protein and extremely recalcitrant biopolymer. β-Keratin is the major constituent of feathers, which, due to the widespread poultry industry, has become a major waste product. Biotechnological upcycling of feather waste has gained interest as various bacteria and fungi capable of degrading keratin have been isolated. These microorganisms produce proteases, termed keratinases, responsible for the enzymatic hydrolysis of keratin. The structural properties that confer keratinolytic activity to proteases are, however, not well understood. Here, we investigated the structure-function relationship of a subtilisin-like S8 endopeptidase (FerB) from the thermophile Fervidobacterium pennivorans strain T. FerB was crystallized and its structure solved to 1.5 Å resolution, revealing an auto-processed state where the pro-peptide domain is non-covalently attached to the catalytic domain. The carboxyl group of the scissile peptide bond is coordinated in the active site within hydrogen bonding distance of the catalytic triad’s serine residue. Unlike fervidolysin, no β-sandwich domains are present. However, a tyrosine-rich β-hairpin structure is found in the corresponding position within the FerB structure. Deletion of the β-hairpin reduced the protein’s integrity and keratinase activity.

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Abstract: Crystallization is a key step in drug purification, offering low cost and facile scalability. The thesis investigates the role of heterogeneous nucleation templates in enhancing crystallization efficiency and controllability with a focus on biopharmaceutical applications, and examines the mechanisms of template-mediated nucleation, crystal growth, and morphology control using carbon-based templates, polymeric hydrogel templates, and microbial bio-templates. The interaction between inorganic salt and proteins was investigated and proteins themselves were also applied as the macromolecular templates. Carbon-based materials, including graphite and graphene oxide (GO), were investigated for their influence on lysozyme crystallization. Graphite reduced nucleation time by 57% compared to those without templates and demonstrated edge adsorption. GO exhibited a nonlinear effect, accelerating nucleation at low lysozyme concentrations (30 mg mL-1) while inhibiting it at higher concentrations (over 50 mg mL-1). Furthermore, a second strategy was pursued using heterogeneous templates based on poly (ethylene glycol) diacrylate (PEGDA) hydrogel microspheres (HMS). In contrast to the adsorption mechanism, the PEGDA HMS acts by releasing precipitant (0- 4 M NaCl) to create localized supersaturation gradients, thereby reducing nucleation time by 79%. Based on the mechanisms of templated crystallization observed in the lysozyme system, this work sought to explore the universality of these effects in inorganic systems critical to biomineralization and disease. The interaction between proteins and inorganic salts was further investigated in two model systems: lithium carbonate (Li'CO') and calcium oxalate (CaOx) with proteins (lysozyme, bovine haemoglobin and mRFP). In both systems, inorganic salt crystals serve as templates that influence subsequent protein adsorption and crystallization, leading to the formation of protein-salt composite crystal structures. Elevated salt concentrations consistently promoted nucleation kinetics. Proteins, however, exhibited complex effects: At low supersaturation, proteins like lysozyme inhibited Li'CO' nucleation by chelating Li'. Conversely, at high supersaturation, proteins self-assemble into oligomers or aggregates, providing additional nucleation sites and accelerating nucleation. In the CaOx system, lysozyme enhanced nucleation across its tested concentration range (0-70 mg mL-1). To bridge our findings on artificial templates to biological contexts, in vivo crystallization is further explored. Inspired by nature, the production of intracellular crystals in Bacillus thuringiensis (Bt) was studied, and its Cry1Ac gene was applied to form a crystal scaffold (CS) as the bio-template to generate crystal nanoparticles in Escherichia coli (E. coli). Through adaptive laboratory evolution (ALE) via serial passaging, we achieved a nearly tenfold increase in protein fluorescence level and produced biologically active nanocrystals with high solubility under alkaline conditions. By integrating heterogeneous nucleation theory with biomimetic strategies, our work elucidates diverse templating mechanisms, including surface, the creation of local supersaturation gradients, and inorganic salt templates. These understandings enable the rational design of templates to control crystallization outcomes. Furthermore, we establish a platform that applying the Cry gene from Bt as the crystal scaffold to function as bio-templates inside cells, demonstrating their potential in high-yield production of bioactive nanocrystals.

Open Access

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Abstract: Extracellular contractile injection systems (eCISs) are phage-derived nanomachines used by bacteria to deliver effectors into target cells. Well-studied examples include the Photorhabdus asymbiotica virulence cassettes and the antifeeding prophage from Serratia entomophila, which have been engineered for heterologous cargo delivery. Recent genomic analyses identified eCIS gene clusters in the opportunistic human pathogen Salmonella enterica subspecies salamae, but their structure, function, and biotechnological potential remain unexplored. Here, we report a high-resolution cryo-electron microscopy structure of the S. enterica eCIS. Our atomic models reveal a distinctive sheath architecture, an expansive cage-like shell around a central spike, and an associated integral membrane protein. We identify a putative effector encoded within the operon exhibiting mild periplasmic toxicity and provide evidence that the S. enterica eCIS deviates from canonical eCISs by interacting with the inner membrane. Guided by these structural features, we uncover, to the best of our knowledge, a previously unannotated cluster of contractile injection systems (CISs). Together, our findings expand the known diversity of CISs’ structures and functions, and lay the groundwork for engineering customisable protein delivery platforms.

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Abstract: We present the structural and functional characterization of a single-stranded DNA-binding protein (SSB-M5) identified from a hot spring metagenome in Vatnajökull National Park, Iceland. This small protein (136 aa; 15,695 Da) shares 100% amino acid sequence identity with two previously uncharacterized SSBs from hyperthermophilic Fervidobacterium species. Functional complementation assay demonstrated that SSB-M5 can substitute for Escherichia coli SSB in an ssb− mutant strain, confirming its biological activity. A recombinant C-terminally His-tagged SSB-M5 was overproduced, purified to homogeneity, and subjected to structural, biochemical, and biophysical analysis. The crystal structure revealed that SSB-M5 forms a dimer through a crystallographic twofold axis, with each monomer contributing to a large antiparallel β-sheet. The flat surfaces of the β-sheets from the two dimers are packed together via a second crystallographic twofold axis, forming a tetramer that serves as the functional unit of the SSB-M5. Electrophoretic mobility shift assays showed that SSB-M5, after heat treatment up to 100°C, forms stable DNA-protein complexes with the (dT)40 oligo. Quantitative analyses revealed that SSB-M5 binds (dT)70 oligonucleotide with very high affinity (KD = 72 ± 6 pM). Hill analysis indicated cooperative binding, yielding an EC50 of 141 pM and a Hill coefficient of 2. Moreover, inclusion of SSB-M5 in PCR reactions significantly enhanced amplification by eliminating non-specific products. Together, these findings identify SSB-M5 as a hyperthermostable, high-affinity single-stranded DNA-binding protein with potential applications in molecular biology and biotechnology.

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Abstract: Enzymes are powerful catalysts for selective transformations but often suffer from limited stability under operational conditions such as elevated temperature or the presence of organic cosolvents. While sequence-based strategies have been widely used to improve stability, chemical protein engineering enables modifications beyond the natural amino acid repertoire thereby offering complementary routes to tailor enzyme function and robustness. Here, we apply the in situ cyclization of proteins (INCYPRO) to a D-stereospecific hydrolase with low intrinsic thermal stability. Site-specific macrocyclization substantially improved resilience to heat and cosolvent stress. Unexpectedly, we discovered a cross-linked protein dimer with enhanced activity and thermal stability. The complex structure was confirmed by x-ray crystallography. Extending the INCYPRO approach, we engineered a multicyclic enzyme dimer with a total of four cross-linking sites, which not only retained high activity under benign conditions but also outperformed the wild-type under stress. Our findings establish protein macrocyclization as a versatile strategy to stabilize both monomeric and multimeric enzymes, providing a powerful route to robust biocatalysts.