Biology subjects

Wilson, R. H.

Publications and source records attributed to Wilson, R. H..

11 recordsLinked to original sources

bioRxiv2026

Machine learning-assisted directed evolution of plant Rubisco

Ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) is foundational to life on Earth, catalyzing carbon dioxide (CO2) fixation to generate biomass. However, Rubisco is a slow and inefficient enzyme that has proven challenging to engineer. We applied the structure-informed machine learning (ML) model ESM-IF1 to identify plausible amino acid sites in the large subunit of Nicotiana tabacum Rubisco to target for directed evolution. ML-assisted library design followed by selection in Rubisco-dependent Escherichia coli identified multiple enriched variants displaying improved catalytic efficiency. Several improved variants carried amino acid changes not found in the evolutionary lineage of plants, despite being assembly competent in plant chloroplasts, demonstrating that ML-assisted protein design can explore functional sequence space beyond what is observed from natural sequence diversity. Most prominently, the T391I substitution improved carboxylation rate by 29% and aerobic carboxylation efficiency by 43%. Our findings demonstrate the utility of ML-assisted evolution for engineering Rubisco with improved carboxylation efficiency and potential for enhancing crop productivity.

plant biology↗

bioRxiv2025

Rational design of tertiary coordination sphere of a heme-based sensor for two-orders enhanced oxygen affinity

Biological O2 sensing is crucial for diverse physiological functions across all forms of life. Heme-containing proteins achieve this by binding O2 to their iron center and have been found to display O2 affinities spanning several orders of magnitude. Despite decades of investigation into the structure and function of heme-based O2 sensors, the molecular mechanisms that enable the tuning of O2 affinity to match specific physiological roles remain unclear. Here, we utilize the O2 sensing mycobacterial DosS protein as a model system to explore the role of heme irons tertiary coordination sphere in controlling its O2 affinity. By rationally and systematically modifying the tertiary coordination sphere to promote the formation of a Trp-Tyr-Asn H-bond triad within the hemes distal pocket, we have enhanced the O2 affinity of WT DosS by over 150-fold. The rationally designed DosS exhibited a Kd value of 3 {+/-} 1 nM, compared to 460 {+/-} 80 nM for WT DosS. Employing a combination of structural, biochemical, spectroscopic, and computational studies, our analysis of WT and designed DosS variants highlights how the interplay between distal H-bond networks and heme-pocket electrostatics drives large differences in their O2 sensing capabilities. Ultimately, our work shows how metalloenzymes can dramatically alter their sensitivity to diatomic signaling molecules by tuning the tertiary coordination sphere, broadly impacting how we understand related biological sensing and signaling.

biochemistry↗

bioRxiv2025

Scaled multidimensional assays of variant effect identify sequence-function relationships in hypertrophic cardiomyopathy

BackgroundAn estimated 1 in 500 people live with hypertrophic cardiomyopathy (HCM), a disease for which genetic diagnosis can identify family members at risk, and increasingly guide therapy. Mutations in the myosin binding protein C3 (MYBPC3) gene account for a significant proportion of HCM cases. However, many of these variants are classified as variants of uncertain significance (VUS), complicating clinical decision-making. Scalable methods for variant interpretation in disease-specific cell types are crucial for understanding variant impact and uncovering disease mechanisms. MethodsWe developed a scaled multidimensional mapping strategy to evaluate the functional impact of variants across a critical domain of MYBPC3. We incorporate saturation base editing at the native MYBPC3 locus, a long-read RNA sequencing-enabled assay of variant splice effects, and measurements of HCM-relevant phenotypes, including MYBPC3 abundance, hypertrophic signaling, and ubiquitin-proteasome function in human induced pluripotent stem cell-derived cardiomyocytes (iPSC-CMs). ResultsOur multidimensional mapping strategy enabled high-resolution functional analysis of MYBPC3 variants in iPSC-CMs. Targeted transient base editing generated a comprehensive variant library at the native locus, capturing diverse variant effects on cellular HCM-relevant phenotypes. Our massively parallel splicing assay identified novel splice-disrupting variants. Integration of functional assays revealed that decreased MYBPC3 abundance is a key driver of HCM-related phenotypes. In parallel, downregulation of protein degradation was observed as a compensatory response to MYBPC3 loss of function, and novel disease mechanisms were identified for missense variants near a critical binding domain, underscoring their contribution to pathogenesis. Bayesian estimates of variant effects enable the reclassification of clinical variants. ConclusionsThis work provides a platform for extending genome engineering in iPSCs to multiplexed assays of variant effects across diverse disease-relevant cellular phenotypes, enhancing the understanding of variant pathogenicity and uncovering novel biological mechanisms that could inform therapeutic strategies.

genetics↗

bioRxiv2025

In vivo directed evolution of an ultra-fast RuBisCO from a semi-anaerobic environment imparts oxygen resistance

Carbon dioxide (CO2) assimilation by the enzyme Ribulose-1,5-bisphosphate Carboxylase/Oxygenase (Rubisco) underpins biomass accumulation in photosynthetic bacteria and eukaryotes. Despite its pivotal role, Rubisco has a slow carboxylation rate [Formula] and is competitively inhibited by oxygen (O2). These traits impose limitations on photosynthetic efficiency, making Rubisco a compelling target for improvement. Interest in Form II Rubisco from Gallionellaceae bacteria, which comprise a dimer or hexamer of large subunits, arises from their nearly 5-fold higher [Formula] than the average Rubisco enzyme. As well as having a fast [Formula] (25.8 s-1 at 25 {degrees}C), we show that Gallionellaceae Rubisco (GWS1B) is extremely sensitive to O2 inhibition, consistent with its evolution under semi-anaerobic environments. We therefore used a novel in vivo mutagenesis-mediated screening pipeline to evolve GWS1B over six rounds under oxygenic selection, identifying three catalytic point mutants with improved ambient carboxylation efficiency; Thr-29-Ala (T29A), Glu-40-Lys (E40K) and Arg-337-Cys (R337C). Full kinetic characterization showed that each substitution enhanced the CO2 affinity of GWS1B under oxygenic conditions by subduing oxygen affinity, leading to 25% (E40K), 11% (T29A) and 8% (R337C) enhancements in carboxylation efficiency under ambient O2 at 25 {degrees}C. By contrast, under the near anaerobic natural environment of Gallionellaceae, the carboxylation efficiency of each mutant was impaired [~]16%. These findings demonstrate the efficacy of artificial directed evolution to access novel regions of catalytic space in Rubisco. SignificanceGiven Rubiscos crucial role in carbon dioxide assimilation, addressing its slow carboxylation rate and oxygen inhibition is a significant challenge. Utilizing one of the fastest known, yet also highly oxygen-sensitive, Rubisco - from the bacteria Gallionellaceae - we applied a novel in vivo directed evolution pipeline in Escherichia coli to discover mutations that specifically enhance carboxylation efficiency under ambient oxygen, a condition distinct from Gallionellaceaes natural semi-anaerobic environment. Our findings underscore the potential of directed evolution to unlock new catalytic capabilities for Rubisco, with implications for both fundamental research and practical agricultural applications.

biochemistry↗

bioRxiv2024

Machine learning guided rational design of a non-heme iron-based lysine dioxygenase improves its total turnover number

Highly selective C-H functionalization remains an ongoing challenge in organic synthetic methodologies. Biocatalysts are robust tools for achieving these difficult chemical transformations. Biocatalyst engineering has often required directed evolution or structure-based rational design campaigns to improve their activities. In recent years, machine learning has been integrated into these workflows to improve the discovery of beneficial enzyme variants. In this work, we combine a structure-based machine-learning algorithm with classical molecular dynamics simulations to down select mutations for rational design of a non-heme iron-dependent lysine dioxygenase, LDO. This approach consistently resulted in functional LDO mutants and circumvents the need for extensive study of mutational activity before-hand. Our rationally designed single mutants purified with up to 2-fold higher yields than WT and displayed higher total turnover numbers (TTN). Combining five such single mutations into a pentamutant variant, LPNYI LDO, leads to a 40% improvement in the TTN (218{+/-}3) as compared to WT LDO (TTN = 160{+/-}2). Overall, this work offers a low-barrier approach for those seeking to synergize machine learning algorithms with pre-existing protein engineering strategies.

biochemistry↗

bioRxiv2024

Controllable multi-halogenation of a non-native substrate by SyrB2 iron halogenase

Geminal, multi-halogenated functional groups are widespread in natural products and pharmaceuticals, yet no synthetic methodologies exist that enable selective multi-halogenation of unactivated C-H bonds. Biocatalysts are powerful tools for late-stage C-H functionalization, as they operate with high degrees of regio-, chemo-, and stereoselectivity. 2-oxoglutarate (2OG)-dependent non-heme iron halogenases chlorinate and brominate aliphatic C-H bonds offering a solution for achieving these challenging transformations. Here, we describe the ability of a non-heme iron halogenase, SyrB2, to controllably halogenate non-native substrate alpha-aminobutyric acid (Aba) to yield mono-chlorinated, di-chlorinated, and tri-chlorinated products. These chemoselective outcomes are achieved by controlling the loading of 2OG cofactor and SyrB2 biocatalyst. By using a ferredoxin-based biological reductant for electron transfer to the catalytic center of SyrB2, we demonstrate order-of-magnitude enhancement in the yield of tri-chlorinated product that were previously inaccessible using any single halogenase enzyme. We also apply these strategies to broaden SyrB2s reactivity scope to include multi-bromination and demonstrate chemoenzymatic conversion of the ethyl side chain in Aba to an ethylyne functional group. We show how steric hindrance induced by the successive addition of halogen atoms on Abas C4 carbon dictates the degree of multi-halogenation by hampering C3-C4 bond rotation within SyrB2s catalytic pocket. Overall, our work showcases the synthetic potential of iron halogenases to facilitate multi-C-H functionalization chemistry.

biochemistry↗

bioRxiv2024

Most human DNA replication initiation is dispersed throughout the genome with only a minority within previously identified initiation zones

BackgroundThe identification of sites of DNA replication initiation in mammalian cells has been challenging. Here, we present unbiased detection of replication initiation events in human cells using BrdU incorporation and single-molecule nanopore sequencing. ResultsIncreases in BrdU incorporation allow us to measure DNA replication dynamics, including identification of replication initiation, fork direction and termination on individual nanopore sequencing reads. Importantly, initiation and termination events are identified on single-molecules with high resolution, throughout S-phase, genome-wide and at high coverage at specific loci using targeted enrichment. We find a significant enrichment of initiation sites within the broad initiation zones identified by population level studies. However, these focused initiation sites only account for [~]20% of all identified replication initiation events. Most initiation events are dispersed throughout the genome and are missed by cell population approaches. This indicates that most initiation occurs at sites that, individually, are rarely used. These dispersed initiation sites contrast with the focused sites identified by population studies, in that they do not show a strong relationship to transcription or a particular epigenetic signature. ConclusionsWe show here that single-molecule sequencing enables unbiased detection and characterisation of DNA replication initiation events, including the numerous dispersed initiation events that replicate most of the human genome.

genomics↗

bioRxiv2023

Electrostatically regulated active site assembly governs reactivity in non-heme iron halogenases

Non-heme iron halogenases (NHFe-Hals) catalyze the direct insertion of a chloride/bromide ion at an unactivated carbon position using a high-valent haloferryl intermediate. Despite more than a decade of structural and mechanistic characterization, how NHFe-Hals preferentially bind specific anions and substrates for C-H functionalization remains unknown. Herein, using lysine halogenating BesD and HalB enzymes as model systems, we demonstrate strong positive cooperativity between anion and substrate binding to the catalytic pocket. Detailed computational investigations indicate that a negatively charged glutamate hydrogen-bonded to irons equatorial-aqua ligand acts as an electrostatic lock preventing both lysine and anion binding in the absence of the other. Using a combination of UV-Vis spectroscopy, binding affinity studies, stopped-flow kinetics investigations, and biochemical assays, we explore the implication of such active site assembly towards chlorination, bromination, and azidation reactivities. Overall, our work highlights previously unknown features regarding how anion-substrate pair binding govern reactivity of iron halogenases that are crucial for engineering next-generation C-H functionalization biocatalysts.

biochemistry↗

bioRxiv2019

Cerebral perfusion and metabolism coupling during a critical time window provides rapid assessment of cardiac arrest severity and prognosis in a preclinical model

Improved quantitative understanding of the dynamic relationship among cerebral blood flow, oxygen consumption, and electrical activity is important to clinicians treating acute brain injury. Such knowledge would elucidate the neurovascular response to ischemia, helping to potentially guide treatment. Using a multimodal optical imaging platform and a clinically-relevant rat model of cardiac arrest (CA) and cardiopulmonary resuscitation (CPR), we continuously measured cerebral blood flow (CBF), brain tissue oxygenation (StO2), cerebral metabolic rate of oxygen (CMRO2), and cerebral electrical activity (electrocorticography; ECoG). Multiple phases of cerebral hemodynamic recovery, with different degrees of mismatch between CBF and CMRO2, were observed following CPR. At 1 min post-resuscitation, we observed that the ratio CBF/CMRO2 is indicative of CA duration/severity and prognostic (with 87% accuracy) of short-term neurological recovery measured by the re-initiation of ECoG activity. These measurements provide the earliest known metrics for assessment of CA severity and prognosis post-CPR. Interestingly, the accuracy of this information is lost beyond 2-3 minutes post-CPR, highlighting a critical, easily overlooked, period immediately post-CPR. These metrics do not require pre-resuscitation data, underscoring translational potential in emergency-response settings when pre-CA information is unavailable. These metrics encourage validation in human studies, potentially offering real-time feedback during CA/CPR to optimize neurological outcome.

neuroscience↗

bioRxiv2019

Spreading depolarization and repolarization during cardiac arrest as an ultra-early marker of neurological recovery in a preclinical model

Spreading depolarization (SD) accompanies numerous neurological conditions, including migraine, stroke, and traumatic brain injury. There is significant interest in understanding the relationship between SD and neuronal injury. However, characteristics underlying SD and repolarization (RP) induced by global cerebral ischemia (e.g., cardiac arrest (CA)) and reperfusion are not well understood. Quantifying features of SD and RP during CA and cardiopulmonary resuscitation (CPR) may provide important metrics for diagnosis and prognosis of neurological injury from hypoxia-ischemia. We characterized SD and RP in a rodent model of asphyxial CA+CPR using a multimodal platform including electrocorticography (ECoG) and optical imaging. We detected SD and RP by (1) alternating current (AC), (2) direct current (DC), and (3) optical imaging of spreading ischemia, spreading edema, and vasoconstriction. Earlier SD (r=-0.80; p<0.001) and earlier RP (r=-0.71, p<0.001) were associated with better neurological recovery after 24hrs. SD+RP onset times predicted good vs poor neurological recovery with 82% sensitivity and 91% specificity. To our knowledge, this is the first preclinical study to link SD and RP characteristics with neurological recovery post-CA. These data suggest that SD and RP may be ultra-early, real-time prognostic markers of post-CA outcome, meriting further investigation into translational implications during global cerebral ischemia.

neuroscience↗

bioRxiv2019

High-speed quantitative optical imaging of absolute metabolism in the rat cortex

Quantitative measures of blood flow and metabolism are essential for improved assessment of brain health and response to ischemic injury. In this report, we demonstrate a multimodal technique for measuring the cerebral metabolic rate of oxygen (CMRO2) in the rodent brain on an absolute scale (M O2 / min). We use laser speckle imaging (LSI) at 809 nm and spatial frequency domain imaging (SFDI) at 655 nm, 730 nm, and 850 nm to obtain spatiotemporal maps of cerebral blood flow (CBF), tissue absorption (a), and tissue scattering (s). Knowledge of these three values enables calculation of a characteristic blood flow speed, which in turn is input to a mathematical model with a \"zero-flow\" boundary condition to calculate absolute CMRO2. We apply this method to a rat model of cardiac arrest (CA) and cardiopulmonary resuscitation. With this model, the zero-flow condition occurs during entry into CA. The CMRO2 values calculated with our method are in good agreement with those measured with magnetic resonance (MR) and positron emission tomography (PET) by other groups. Our technique provides a quantitative metric of cerebral metabolism that can potentially be used for comparison between animals and longitudinal monitoring of a single animal over multiple days, to assess differences in baseline metabolism and track recovery of metabolism in survival studies following ischemia and reperfusion.

neuroscience↗