Ajasja Ljubetič

Filamentous bacteriophages are monodisperse, high-aspect-ratio protein nanofibers that provide a scalable and genetically addressable platform for biohybrid nanomaterials. However, direct fusion of heterologous proteins to capsid proteins often disrupts assembly and limits high-density functionalization to short peptides. Here, we report a dual-display “type 388” phage vector enabling orthogonal and adjustable surface functionalization. SnoopTag was fused to the minor coat protein p3 for low-valency tip display, while SpyTag variants were displayed on the major coat protein p8 for sidewall presentation. Using fluorescent and immunochemical methods, and high-resolution microscopy techniques, we show that the lateral display density on p8 can be discretely adjusted by selecting distinct signal sequence-SpyTag combinations that modulate the efficiency of bacterial inner membrane integration and capsid incorporation. Replacement of the native p8 signal sequence with a signal recognition particle-compatible counterpart enhances functional display, enabling adjustable multivalent patterning. Orthogonal Tag/Catcher pairs and post-assembly cargo conjugation together establish a plug-and-play, anisotropic nanoscaffold for precision biofunctionalization. As a proof of concept, the phage nanotemplate was functionalized with targeting and reporter modules to generate an antibody-mimetic probe for analyte detection.

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Over the past decade, protein design has evolved from a specialized discipline into a broadly accessible approach for engineering and interrogating biological systems. Despite these advances, protein design continues to be a technically challenging task, often requiring knowledge of programming to be able to use and combine the different software packages. To address this challenge, we have developed Prosculpt, an easy-to-use protein design pipeline. Prosculpt integrates RFdiffusion for backbone generation, ProteinMPNN for sequence design and multiple structure-prediction platforms (AF2, AF3, Colabfold, Boltz2). Candidate designs are evaluated using customizable Rosetta-based scoring protocols. Each project is specified through a single configuration file, enabling users with minimal computational expertise to perform sophisticated protein design tasks without writing code, while also allowing advanced users to access the full capabilities of the underlying programs. Prosculpt supports a wide range of applications, including design of symmetric homo-oligomers, design of binders, motif scaffolding, partial diffusion and fixed-backbone sequence redesign. By combining these capabilities within a single, user-friendly platform, Prosculpt provides a practical entry point to modern protein design for both novice and expert users.

Molecular machines hold great potential. Design of static monomeric and oligomeric protein structures has advanced tremendously, but few dynamic protein systems have been designed. Here we present the design and characterization of a random protein walker that diffuses along a designed protein track. For the track, we designed micro-meter long fibres and developed a method to rigidly decorate them with arbitrary proteins. The walkers consist of homo-oligomers that reversibly bind the track using heterodimeric feet; we tested multiple heterodimer interfaces for reversibility and designed six walkers with different numbers of feet. Cryo-EM experiments confirmed the structure of the track and walkers. We performed detailed single molecule tracking and kinetics characterisation and found that the walkers with more feet diffuse along the track faster. The system represents a tuneable starting point for future powered protein molecular robots.

Versatile DNA and polypeptide‐based structures have been designed based on complementary modules. However, polypeptides can also form higher oligomeric states. We investigated the introduction of tetrameric modules as a substitute for coiled‐coil dimerization units used in previous modular nanostructures. Tetramerizing helical bundles can run in parallel or antiparallel orientation, expanding the number of topological solutions for modular nanostructures. Furthermore, this strategy facilitates the construction of nanostructures from two identical polypeptide chains. Importantly, tetrameric modules substantially stabilized protein nanostructures against air–water interface denaturation, enabling the determination of the first cryo‐electron microscopy three‐dimensional structure of a coiled‐coil‐based nanostructure, confirming the designed agreement of the modules forming a tetrahedral cage.

Versatile DNA and polypeptide‐based structures have been designed based on complementary modules. However, polypeptides can also form higher oligomeric states. We investigated the introduction of tetrameric modules as a substitute for coiled‐coil dimerization units used in previous modular nanostructures. Tetramerizing helical bundles can run in parallel or antiparallel orientation, expanding the number of topological solutions for modular nanostructures. Furthermore, this strategy facilitates the construction of nanostructures from two identical polypeptide chains. Importantly, tetrameric modules substantially stabilized protein nanostructures against air–water interface denaturation, enabling the determination of the first cryo‐electron microscopy three‐dimensional structure of a coiled‐coil‐based nanostructure, confirming the designed agreement of the modules forming a tetrahedral cage.

Protein–protein interactions (PPIs) regulate many cellular processes and engineered PPIs have cell and gene therapy applications. Here, we introduce massively parallel PPI measurement by sequencing (MP3-seq), an easy-to-use and highly scalable yeast two-hybrid approach for measuring PPIs. In MP3-seq, DNA barcodes are associated with specific protein pairs and barcode enrichment can be read by sequencing to provide a direct measure of interaction strength. We show that MP3-seq is highly quantitative and scales to over 100,000 interactions. We apply MP3-seq to characterize interactions between families of rationally designed heterodimers and to investigate elements conferring specificity to coiled-coil interactions. Lastly, we predict coiled heterodimer structures using AlphaFold-Multimer (AF-M) and train linear models on physics-based energy terms to predict MP3-seq values. We find that AF-M-based models could be valuable for prescreening interactions but experimentally measuring interactions remains necessary to rank their strengths quantitatively.

Protein-protein interactions (PPIs) are crucial for biological functions and have applications ranging from drug design to synthetic cell circuits. Coiled-coils have been used as a model to study the sequence determinants of specificity. However, building well-behaved sets of orthogonal pairs of coiled-coils remains challenging due to inaccurate predictions of orthogonality and difficulties in testing at scale. To address this, we develop the next-generation bacterial two-hybrid (NGB2H) method, which allows for the rapid exploration of interactions of programmed protein libraries in a quantitative and scalable way using next-generation sequencing readout. We design, build, and test large sets of orthogonal synthetic coiled-coils, assayed over 8,000 PPIs, and used the dataset to train a more accurate coiled-coil scoring algorithm (iCipa). After characterizing nearly 18,000 new PPIs, we identify to the best of our knowledge the largest set of orthogonal coiled-coils to date, with fifteen on-target interactions. Our approach provides a powerful tool for the design of orthogonal PPIs.

New variants of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) continue to arise and prolong the coronavirus disease 2019 (COVID-19) pandemic. Here, we used a cell-free expression workflow to rapidly screen and optimize constructs containing multiple computationally designed miniprotein inhibitors of SARS-CoV-2. We found the broadest efficacy was achieved with a homotrimeric version of the 75-residue angiotensin-converting enzyme 2 (ACE2) mimic AHB2 (TRI2-2) designed to geometrically match the trimeric spike architecture. Consistent with the design model, in the cryo-electron microscopy structure TRI2-2 forms a tripod at the apex of the spike protein that engaged all three receptor binding domains simultaneously. TRI2-2 neutralized Omicron (B.1.1.529), Delta (B.1.617.2), and all other variants tested with greater potency than the monoclonal antibodies used clinically for the treatment of COVID-19. TRI2-2 also conferred prophylactic and therapeutic protection against SARS-CoV-2 challenge when administered intranasally in mice. Designed miniprotein receptor mimics geometrically arrayed to match pathogen receptor binding sites could be a widely applicable antiviral therapeutic strategy with advantages over antibodies in greater resistance to viral escape and antigenic drift, and advantages over native receptor traps in lower chances of autoimmune responses.