Biology subjects

Brackenbrough, E.

Publications and source records attributed to Brackenbrough, E..

7 recordsLinked to original sources

bioRxiv2025

Inhibition of ice recrystallization with designed twistless helical repeat proteins

Given the repetitive structure of crystalline ice, its unsurprising that highly active ice-binding proteins, often with beta-roll structures, also have repeating motifs. Here, we introduce a de novo designed family of ice-binding twistless alpha-helical repeat (iTHR) proteins. Each iTHR protein comprises two planar layers of parallel alpha-helices connected by loops--a structural topology not seen in native ice-binding proteins. The ice-binding helices feature an ordered array of TXXXAXXXAXX motifs, spaced to match the pyramidal {201} and secondary prism plane {110} ice lattice facets, and engineered with a 98.2{degrees} turn angle per residue to orient threonines uniformly towards the ice surface. iTHR proteins show high solubility, thermostability, and produce varied ice crystal morphologies depending on their intended target facet. Crucially, iTHRs exhibit ice recrystallization inhibition (IRI) at critical concentrations comparable to those of many native globular ice-binding proteins. Extensive site-specific mutagenesis shows that ice-binding activity in iTHR proteins is robust, remaining largely unaffected by changes in chemical composition. Variation in the repeat number reveals a non-monotonic relationship to IRI activity. X-ray crystal structures of two designs confirm the intended orientation of threonines, uniformly pointing toward the ice surface. The iTHR family provides a versatile platform to systematically investigate the complex structure-activity relationships underlying protein-ice interactions.

biophysics↗

bioRxiv2025

Massively parallel assessment of designed protein solution properties using mass spectrometry and peptide barcoding

Library screening and selection methods can determine the binding activities of individual members of large protein libraries given a physical link between protein and nucleotide sequence, which enables identification of functional molecules by DNA sequencing. However, the solution properties of individual protein molecules cannot be probed using such approaches because they are completely altered by DNA attachment. Mass spectrometry enables parallel evaluation of protein properties amenable to physical fractionation such as solubility and oligomeric state, but current approaches are limited to libraries of 1,000 or fewer proteins. Here, we improved mass spectrometry barcoding by co-synthesizing proteins with barcodes optimized to be highly multiplexable and minimally perturbative, scaling to libraries of >5,000 proteins. We use these barcodes together with mass spectrometry to assay the solution behavior of libraries of de novo-designed monomeric scaffolds, oligomers, binding proteins and nanocages, rapidly identifying design failure modes and successes.

biochemistry↗

bioRxiv2024

Accurate de novo design of high-affinity protein binding macrocycles using deep learning

The development of macrocyclic binders to therapeutic proteins typically relies on large-scale screening methods that are resource-intensive and provide little control over binding mode. Despite considerable progress in physics-based methods for peptide design and deep-learning methods for protein design, there are currently no robust approaches for de novo design of protein-binding macrocycles. Here, we introduce RFpeptides, a denoising diffusion-based pipeline for designing macrocyclic peptide binders against protein targets of interest. We test 20 or fewer designed macrocycles against each of four diverse proteins and obtain medium to high-affinity binders against all selected targets. Designs against MCL1 and MDM2 demonstrate KD between 1-10 M, and the best anti-GABARAP macrocycle binds with a KD of 6 nM and a sub-nanomolar IC50 in vitro. For one of the targets, RbtA, we obtain a high-affinity binder with KD < 10 nM despite starting from the target sequence alone due to the lack of an experimentally determined target structure. X-ray structures determined for macrocycle-bound MCL1, GABARAP, and RbtA complexes match very closely with the computational design models, with three out of the four structures demonstrating Ca RMSD of less than 1.5 [A] to the design models. In contrast to library screening approaches for which determining binding mode can be a major bottleneck, the binding modes of RFpeptides-generated macrocycles are known by design, which should greatly facilitate downstream optimization. RFpeptides thus provides a powerful framework for rapid and custom design of macrocyclic peptides for diagnostic and therapeutic applications.

biochemistry↗

bioRxiv2024

Design of facilitated dissociation enables control over cytokine signaling duration

Protein design has focused primarily on the design of ground states, ensuring they are sufficiently low energy to be highly populated1. Designing the kinetics and dynamics of a system requires, in addition, the design of excited states that are traversed in transitions from one low-lying state to another2,3. This is a challenging task as such states must be sufficiently strained to be poorly populated, but not so strained that they are not populated at all, and because protein design methods have generally focused on creating near-ideal structures4-7. Here we describe a general approach for designing systems which use an induced-fit power stroke8 to generate a structurally frustrated9 and strained excited state, allosterically driving protein complex dissociation. X-ray crystallography, double electron-electron resonance spectroscopy, and kinetic binding measurements demonstrate that incorporating excited states enables design of effector-induced increases in dissociation rates as high as 6000-fold. We highlight the power of this approach by designing cytokine mimics which can be dissociated within seconds from their receptors.

biochemistry↗

bioRxiv2024

Computational design of serine hydrolases

Enzymes that proceed through multistep reaction mechanisms often utilize complex, polar active sites positioned with sub-angstrom precision to mediate distinct chemical steps, which makes their de novo construction extremely challenging. We sought to overcome this challenge using the classic catalytic triad and oxyanion hole of serine hydrolases as a model system. We used RFdiffusion1 to generate proteins housing catalytic sites of increasing complexity and varying geometry, and a newly developed ensemble generation method called ChemNet to assess active site geometry and preorganization at each step of the reaction. Experimental characterization revealed novel serine hydrolases that catalyze ester hydrolysis with catalytic efficiencies (kcat/Km) up to 3.8 x 103 M-1 s-1, closely match the design models (C RMSDs < 1 [A]), and have folds distinct from natural serine hydrolases. In silico selection of designs based on active site preorganization across the reaction coordinate considerably increased success rates, enabling identification of new catalysts in screens of as few as 20 designs. Our de novo buildup approach provides insight into the geometric determinants of catalysis that complements what can be obtained from structural and mutational studies of native enzymes (in which catalytic group geometry and active site makeup cannot be so systematically varied), and provides a roadmap for the design of industrially relevant serine hydrolases and, more generally, for designing complex enzymes that catalyze multi-step transformations.

biochemistry↗

bioRxiv2024

Diffusing protein binders to intrinsically disordered proteins

Proteins which bind intrinsically disordered proteins (IDPs) and intrinsically disordered regions (IDRs) with high affinity and specificity could have considerable utility for therapeutic and diagnostic applications. However, a general methodology for targeting IDPs/IDRs has yet to be developed. Here, we show that starting only from the target sequence of the input, and freely sampling both target and binding protein conformation, RFdiffusion can generate binders to IDPs and IDRs in a wide range of conformations. We use this approach to generate binders to the IDPs Amylin, C-peptide and VP48 in a range of conformations with Kds in the 3 -100nM range. The Amylin binder inhibits amyloid fibril formation and dissociates existing fibers, and enables enrichment of amylin for mass spectrometry-based detection. For the IDRs G3bp1, common gamma chain (IL2RG) and prion, we diffused binders to beta strand conformations of the targets, obtaining 10 to 100 nM affinity. The IL2RG binder colocalizes with the receptor in cells, enabling new approaches to modulating IL2 signaling. Our approach should be widely useful for creating binders to flexible IDPs/IDRs spanning a wide range of intrinsic conformational preferences.

biochemistry↗

bioRxiv2024

Controlling semiconductor growth with structured de novo protein interfaces

Protein design now enables the precise arrangement of atoms on the nanometer length scales of inorganic crystal nuclei, opening up the possibility of templating the growth of metal oxides including semiconductors. We designed proteins presenting regularly repeating interfaces containing functional groups that organize ions and water molecules, and characterized their ability to bind to and template metal oxides. Two interfaces promoted the growth of hematite under conditions that otherwise resulted in the formation of magnetite. Three interfaces promoted ZnO nucleation under conditions where traditional ZnO-binding peptides and control proteins were ineffective. Designed cyclic assemblies with these ZnO nucleating interfaces lining interior cavities promoted ZnO growth within the cavity. CryoEM analysis of a designed octahedral nanocage revealed atomic density likely corresponding to the growing ZnO directly adjacent to the designed nucleation promoting interfaces. These findings demonstrate that designed proteins can direct the formation of metal oxides not observed in biological systems, opening the door to protein-semiconductor hybrid materials. One Sentence SummaryWe describe the design of structured protein interfaces that bind to, promote, and localize the growth of zinc oxide and hematite, inorganic materials which are not found in biological systems.

bioengineering↗