Christopher Williams

Designing redox proteins with predictable and tunable electron transfer properties is a major goal in de novo bioenergetics. Here we show that replacing heme B with a series of structurally conservative non-natural metalloporphyrins enables broad modulation of redox potentials over 400 mV in the de novo designed monoheme m4D2 and diheme 4D2 T19D. The non-natural porphyrins bind with high affinity and do not compromise either the heme binding site or global protein structure, as evidenced by X-ray crystallography and NMR spectroscopy. We also report the native-like NMR structure of m4D2 loaded with the non-natural and symmetric iron 2,4-dimethyldeuteroporphyrin IX, confirming our modular approach to tetrahelical redox protein design. This work establishes a versatile platform for constructing tunable electron carriers for engineered bioenergetic pathways and bioelectronic applications.

Designing photochemical properties in proteins is a frontier challenge in biomolecular design, which, when realised, will open new avenues in catalysis and biomolecular electronics. Here we demonstrate an integrated computational design framework, supported by ultrafast spectroscopy, combining protein design, quantum mechanical/molecular mechanics molecular dynamics (QM/MM MD) and electronic structure calculations, to control flavin excited states in de novo proteins. Iterative design yields FC-4D2, a flavocytochrome with covalently bound flavin and non-covalently bound heme B and Fl-4D2, where the heme-binding site is removed. Both proteins are highly thermostable, and access all physiologically relevant flavin redox and protonation states, while high-resolution X-ray crystallography confirms the near-Ångström-level accuracy in design. Incorporating QM/MM MD and linear-response time-dependent density functional theory (LR-TDDFT) into the design workflow enables engineering of a flavin–tryptophan charge-transfer variant, Fl-4D2 _CT , that modifies the flavin absorption spectrum, substantially alters its redox properties and suppresses fluorescence. Ultrafast studies reveal how protein design reshapes the excited state relaxation timescale and mechanism. Our study establishes a modular strategy for programming flavin photochemistry in designed proteins.

Designing redox proteins with predictable and tuneable electron transfer properties is a major goal in de novo bioenergetics. Here we show that replacing heme B with a series of structurally conservative non natural metalloporphyrins enables broad modulation of redox potentials over 400 mV in the de novo designed monoheme m4D2 and diheme 4D2 T19D. The non-natural porphyrins bind with high affinity and do not compromise either the heme binding site or global protein structure, as evidenced by X-ray crystallography and NMR spectroscopy. We also report the native-like NMR structure of m4D2 loaded with the non-natural and symmetric iron 2,4-dimethyldeuteroporphyrin IX, confirming our modular approach to tetrahelical redox protein design. This work establishes a versatile platform for constructing tuneable electron carriers for engineered bioenergetic pathways and bioelectronic applications.

Phosphorylation at serine 129 (pS129) is a dominant post-translational modification of α-synuclein (αSyn) and a widely used pathological marker in Parkinson's disease, yet its mechanistic consequences remain debated across physiological and pathological contexts. Most studies rely on phosphomimetic substitutions such as S129D, which approximate net charge but do not reproduce the steric, geometric, or hydrogen-bonding properties of authentic phosphorylation. Here, we establish a robust bacterial coexpression platform that generates homogeneous, site-specifically phosphorylated αSyn using its native kinase, Polo-like kinase 2. Using this system, we show that authentic pS129 differs fundamentally from S129D: it induces local, NMR-detectable perturbations within the C-terminal conformational ensemble, exhibits distinct and context-dependent aggregation behavior, and elicits neuronal responses and modest, reproducible toxicity not reproduced by phosphomimetics. These data resolve inconsistencies in the literature and highlight the importance of chemically authentic post-translational modification. More broadly, this platform provides a generalizable and scalable route to chemically faithful phosphorylated proteins, enabling more accurate interrogation of post-translationally regulated protein function.

Nuclear Transcription Factor-Y (NF-Y) is a transcription factor that binds CCAAT motifs to regulate gene expression, controlling cell proliferation, metabolism, and differentiation. NF-Y dysregulation contributes to diverse pathologies, including cancer, neurological disorders, cardiovascular disease, and tissue fibrosis. Using in silico molecular docking, we screened a library of eight million compounds to identify molecules targeting a pocket on the NF-YB/NF-YC dimer. We identified one compound, designated NFYi5, that was able to reduce the NF-Y activity. NFYi5 reduced mRNA levels of NF-Y target genes, while sparing housekeeping gene expression, and inhibiting cell proliferation. Mechanistic studies revealed that NFYi5 impaired NF-Y–DNA binding and accelerated NF-YA protein degradation, reducing its half-life from 16.5 ± 1.5 h to 8.5 ± 0.7 h. Together, these data establish NFYi5 as a small-molecule that can reduce NF-Y activity and is associated with antimitogenic properties. This proof-of-concept study demonstrates that NF-Y is pharmacologically tractable and highlights NFYi5 as a potential lead compound for therapeutic development in NF-Y-driven diseases.

Indoleamine 2,3-dioxygenase (IDO) is a heme-dependent enzyme that catalyzes the first, rate-limiting step of the kynurenine pathway─the oxidation of l-tryptophan to N-formylkynurenine (NFK). IDO-catalyzed depletion of tryptophan levels and accumulation of kynurenine pathway metabolites is an important control mechanism of the immune responses in cells. IDO has been considered as a dioxygenase because two atoms of oxygen are inserted into the substrate. Here, we use LC-MS and NMR to examine the reactivity of human IDO (hIDO) with l-tryptophan (l-Trp) and several other tryptophan analogues. Alongside dioxygenase activity, we identify a concurrent pathway of heme-dependent monooxygenase activity in the reaction of hIDO with l-Trp, leading to the formation of a cyclic 3a-hydroxy-1,2,3,3a,8,8a-hexahydropyrrolo[2,3-b]indole-2-carboxylic acid (HPIC) species. Reaction profiles for the reaction of hIDO with other tryptophan analogues are likewise examined. Formation of HPIC from l-Trp is reproduced in HeLa cells induced to overexpress hIDO, indicating that this dual dioxygenase/monooxygenase reactivity also occurs biologically. Notably, the reaction of hIDO with β-[3-benzo(b)thienyl]-l-alanine (S-l-Trp)─a known inhibitor ─yielded only the cyclic HPIC analogue, suggesting that IDO activity can be selectively directed toward the monooxygenase pathway. Molecular dynamics simulations underscore the critical role of substrate plasticity within the active site of hIDO, while DFT calculations provide a mechanistic rationalization for the observed product distributions. Together, the data demonstrate dual dioxygenase/monooxygenase functionality for human IDO. As the overall gatekeeper for control of tryptophan levels in cells, the findings provide mechanistic information on relevance to therapeutic strategies focused on IDO inhibition.

Bongkrekic acid biosynthesis requires a cassette of β-branching enzymes (BonF–BonI) to install two distinct β-branches. Here we confirm the roles of BonF–BonI in vitro and uncover a previously unannotated ACP _D , BonN.

Preventing the aggregation of α-synuclein (αS) into toxic oligomers and conformers is a major therapeutic goal in conditions such as Parkinson’s disease and Lewy body dementia. However, the large intracellular protein–protein interfaces within such aggregates make this a challenging target for small molecule approaches or biologics, which often lack cell permeability. Peptides occupy a suitable middle ground and are increasingly being explored as preventative treatments. We previously showed that the N-terminal lipid binding region (αS_1–25) inhibits αS aggregation. Building on this, we designed a series of N- and C-terminal truncations to systematically reduce the peptide length, enabling a 56% downsizing (i.e., truncating 92% of the full-length αS protein), to identify the smallest functional unit capable of binding αS and potently blocking its aggregation and toxicity. We next introduced seven systematic i → i + 4 helix constraints to assess impact on (i) α-helicity, (ii) aggregation inhibition, (iii) serum stability, (iv) neuronal uptake, and (v) phenotypic rescue. This work maps key amphipathic features and identifies residues that are critical for αS engagement and inhibitory activity. The most effective helix-constrained peptide, αS_2–12(L6), showed marked improvements across all metrics and represents a strong candidate for further therapeutic development.

The leinamycin family of polyketides are promising antitumor antibiotics, yet several aspects of their biosynthesis remain elusive. All leinamycin family members bear a sulfur‐containing moiety which is essential for the anticancer activity exhibited by leinamycin. The key building blocks required for the incorporation of these functionalities are introduced in the final module of the polyketide synthase (PKS), which elegantly combines β‐branching and thiocysteine incorporation to generate a diverse library of sulfur‐based molecular scaffolds. Two acyl carrier proteins (ACPs) form a key didomain component of this module, but their amino acid sequence divergence has brought into question the common notion of functional equivalence. Here, we provide unprecedented functional evidence that these tandem ACPs play distinct roles in the final module of polyketide assembly. Using the weishanmycin biosynthetic pathway as a template, the in vitro reconstitution of key polyketide chain extension and β‐branching steps in this module has revealed strict functional selectivity for a single ACP. Furthermore, we propose a cryptic transacylation step must occur prior to polyketide off‐loading and cyclization. Altogether, these mechanistic investigations suggest that an atypical in‐series mechanism underpins sulfur incorporation in the leinamycin family, and provides significant progress towards delineating their late‐stage assembly.