Anthony A. Hyman

Transcription factors (TFs) regulate gene expression by binding to specific genomic loci determined by DNA sequence. Their sequence specificity is commonly summarized by a consensus binding motif. However, eukaryotic genomes contain billions of low-affinity DNA sequences to which TFs associate with a sequence-dependent binding energy. We currently lack insight into how the genomic sequence defines this spectrum of binding energies and the resulting pattern of TF localization. Here, we set out to obtain a quantitative understanding of sequence-dependent TF binding to both motif and nonmotif sequences. We achieve this by first pursuing accurate measurements of physical binding energies of human TF Krüppel-like factor 4 (Klf4) to a library of short DNA sequences in a fluorescence-anisotropy-based bulk competitive binding assay. Second, we show that the highly nonlinear sequence dependence of Klf4 binding energies can be captured by combining a linear model of binding energies with an Ising model of the coupled recognition of nucleotides by a TF. In this model, nucleotide–TF interfaces collectively switch between two states, weak binding and strong nucleotide-specific binding, and the states of neighboring interfaces are coupled with J ∼ 2 k B T . We find that this statistical mechanics model parameterized by our in vitro measurements captures Klf4 binding patterns on an individual long DNA molecule stretched in the optical tweezer, and is predictive for Klf4 occupancy across the entire human genome without additional fit parameters.

Mitotic centrosome maturation requires Polo/PLK1-dependent expansion of the pericentriolar material (PCM). In Drosophila , Centrosomin (Cnn) assembles a scaffold around mitotic centrioles through interactions between its PReM and CM2 domains. Here, we show that PReM adopts an autoinhibited helical hairpin conformation that prevents CM2 binding. Polo/PLK1 phosphorylation relieves this autoinhibition, enabling scaffold assembly, whereas phospho-blocking mutations disrupt PReM–CM2 binding in vitro and Cnn scaffold assembly in vivo. Potential functionally analogous domains have been identified in the human and C. elegans Cnn homologues CDK5RAP2 and SPD-5. We find that the human protein appears to share a structurally similar mechanism for scaffold assembly, but the worm protein does not. Consistent with this, deletion of these domains alters the dynamics of Cnn condensates in vitro, but has little effect on SPD-5 condensate dynamics. We conclude that Polo/PLK1 promotes mitotic centrosome assembly, at least in part, by relieving autoinhibitory intramolecular interactions.

How inherent redox activity arises in biomolecular condensates remains unclear. Unlike interfacial systems, such as water microdroplets, where water oxidation underpins redox chemistry, condensates comprise biomolecules that can potentially furnish alternative electron-transfer routes. Here, using electron paramagnetic resonance, electrochemical potentiometry, mass spectrometry, and confocal microscopy assays, we discovered that orthogonal to water oxidation, microenvironment-dependent spontaneous tyrosine oxidation encodes an alternative redox pathway. Through proton-coupled electron transfer, self-induced tyrosine autoxidation in condensates drives the formation of reactive carbon and oxygen species, providing a pathway in parallel to hydroxide oxidation for hydrogen peroxide formation in condensates. This self-induced redox pathway modulates nonequilibrium condensate behaviors, including responses to external chemical perturbations and evolution of the condensate interior microenvironment. By correlating condensate biomolecular composition with inherent redox activities, our work establishes a conceptual framework suggesting that condensate-dependent electron transfer can be critical to define the functions of condensates and deliver a new redox mechanism for cell biology.

Electrochemical gradients are essential to the functioning of cells and form across membranes using active transporters. Here we show in contrast that condensed biomolecular systems—often termed condensates—sustain pH gradients without any external energy input. By studying individual condensates on the micrometre scale using a microdroplet platform, we reveal dense-phase pH shifts towards conditions of minimal electrostatic repulsion. We demonstrate that protein condensates can drive substantial alkaline and acidic gradients, which are compositionally tunable and can extend to complex architectures sustaining multiple unique pH conditions simultaneously. Through in silico characterization of human proteomic condensate networks, we further highlight potential wide-ranging electrochemical properties emerging from condensation in nature, while correlating intracellular condensate pH gradients with complex biomolecular composition. Together, the emergent nature of condensation shapes distinct pH microenvironments, thereby creating a regulatory mechanism to modulate biochemical activity in living and artificial systems.

De novo design of enzymes that cleave amide bonds represents a major challenge ^1–2 . Here we use RFdiffusion methods ^3–4 to de novo design proteins with water activating zinc based active sites adjacent to a peptide substrate binding pocket. Of 135 designs tested, 36% catalyze cleavage of their cognate peptide substrates at the targeted scissile bond. The best design accelerates peptide-bond hydrolysis by more than 10 ⁸ -fold relative to the uncatalyzed reaction; four substitutions increase this acceleration to over 10 ¹⁰ -fold by improving active-site preorganization ⁵ . We use this approach to design metalloproteases that selectively cleave the human disease associated proteins TDP-43, amyloid-β, and serum amyloid A, with rate enhancements of up to 10 ⁹ -fold. We further design caged cytokines and receptor antagonists that are selectively activated by the designed proteases, enabling bioorthogonal control of cell state. Thus, de novo design can yield highly active, programmable proteases with applications in therapeutics, biotechnology, and bioremediation.

Centrosomal material assembles rapidly in mitosis. In Drosophila , the coiled-coil protein Cnn forms a scaffold that recruits PCM clients; in C.elegans , SPD-5 plays an analogous role. Here we show that full-length Cnn and SPD-5 can both form spherical condensates in vitro , but that the interactions driving their assembly into scaffolds inside cells appear to diverge. We show that the Cnn PReM adopts a helical hairpin fold that autoinhibits CM2 binding but that phosphorylation appears to increase hairpin breathing to permit CM2 engagement and robust scaffold assembly. Phospho-blocking mutations prevent PReM–CM2 interactions and scaffold formation, whereas phospho-mimetic substitutions partially restore function. The human homologue CDK5RAP2 contains a CM2 domain that can partially substitute for fly CM2 in vivo and we identify a candidate CDK5RAP2 PReM region that forms macromolecular networks with human CM2 in vitro . By contrast, the putative PReM and CM2 regions of SPD-5 cannot substitute for their equivalent fly domains and they do not interact detectably, suggesting a distinct assembly mechanism in worms despite conserved PLK1-dependent control of PCM growth.

Biomolecular condensates provide compartments that organize biological processes in the cell. Spatial organization of condensates relies on transport across phase boundaries. We investigate the kinetics of molecule transport across interfaces in phase-separated mixtures. Using non-equilibrium thermodynamics we derive the interfacial kinetics, describing the movement of the interface, and of molecular transport across the interface. In the limit of a thin interface, we show that breaking local equilibrium at the interface introduces a local transport resistance. Subsequently, using a continuum approach, we explore two physical scenarios where such a resistance emerges: a mobility minimum and a potential barrier at the interface. These scenarios lead to the same effective transport model in the sharp interface limit, characterized by a single interface resistance parameter. We discuss experimental conditions for which interface resistance could be observed and how these scenarios could be distinguished.

Many subcellular compartments are biomolecular condensates made of multiple components, often including several distinct proteins and nucleic acids. However, current tools to measure condensate composition are limited and cannot capture this complexity quantitatively because they either require fluorescent labels, which can perturb composition, or can distinguish only one or two components. Here we describe a label-free method based on quantitative phase imaging and analysis of tie-lines and refractive index to measure the composition of reconstituted condensates with multiple components. We first validate the method empirically in binary mixtures, revealing sequence-encoded density variation and complex ageing dynamics for condensates composed of full-length proteins. We then use analysis of tie-lines and refractive index to simultaneously resolve the concentrations of five macromolecular solutes in multicomponent condensates containing RNA and constructs of multiple RNA-binding proteins. Our measurements reveal an unexpected decoupling of density and composition, highlighting the need to determine molecular stoichiometry in multicomponent condensates. We foresee this approach enabling the study of compositional regulation of condensate properties and function.

Biomolecular phase separation is an emerging theme for protein assembly and cellular organisation. The collective forces driving such condensation, however, remain challenging to characterise. Here we show that tracking the dilute phase concentration of only one component suffices to quantify composition and energetics of multicomponent condensates. Applying this assay to several disease- and stress-related proteins, we find that monovalent ions can either deplete from or enrich within the dense phase in a context-dependent manner. By analysing the effect of the widely used modulator 1,6-hexanediol, we find that the compound inhibits phase separation by acting as a solvation agent that expands polypeptide chains. Extending the strategy to in cellulo data, we even quantify the relative energetic contributions of individual proteins within complex condensates. Together, our approach provides a generic and broadly applicable tool for dissecting the forces governing biomolecular condensation and guiding the rational modulation of condensate behaviour.