Biology subjects

Walsh, M. E.

Publications and source records attributed to Walsh, M. E..

4 recordsLinked to original sources

bioRxiv2023

Designing Efficient Enzymes: Eight Predicted Mutations Convert a Hydroxynitrile Lyase into an Efficient Esterase

Hydroxynitrile lyase from rubber tree (HbHNL) shares 45% identical amino acid residues with the homologous esterase from tobacco, SABP2, but the two enzymes catalyze different reactions. The x-ray structures reveal a serine-histidine-aspartate catalytic triad in both enzymes along with several differing amino acid residues within the active site. Previous exchange of three amino acid residues in the active site of HbHNL with the corresponding amino acid residue in SABP2 (T11G-E79H-K236M) created variant HNL3, which showed low esterase activity toward p-nitrophenyl acetate. Further structure comparison reveals additional differences surrounding the active site. HbHNL contains an improperly positioned oxyanion hole residue and differing solvation of the catalytic aspartate. We hypothesized that correcting these structural differences would impart good esterase activity on the corresponding HbHNL variant. To predict the amino acid substitutions needed to correct the structure, we calculated shortest path maps for both HbHNL and SABP2, which reveal correlated movements of amino acids in the two enzymes. Replacing four amino acid residues (C81L-N104T-V106F-G176S) whose movements are connected to the movements of the catalytic residues yielded variant HNL7TV (stabilizing substitution H103V was also added), which showed an esterase catalytic efficiency comparable to that of SABP2. The x-ray structure of an intermediate variant, HNL6V, showed an altered solvation of the catalytic aspartate and a partially corrected oxyanion hole. This dramatic increase in catalytic efficiency demonstrates the ability of shortest path maps to predict which residues outside the active site contribute to catalytic activity.

bioengineering↗

bioRxiv2022

Machine Learning Optimization of Candidate Antibodies Yields Highly Diverse Sub-nanomolar Affinity Antibody Libraries

Therapeutic antibodies are an important and rapidly growing drug modality. However, the design and discovery of early-stage antibody therapeutics remain a time and cost-intensive endeavor. In this work, we present an end-to-end Bayesian, language model-based method for designing large and diverse libraries of high-affinity single-chain variable fragments (scFvs). We integrate target-specific binding affinities with information from millions of natural protein sequences in a probabilistic machine learning framework to design thousands of scFvs that are then empirically measured. In a head-to-head comparison with a directed evolution approach, we show that the best scFv generated from our method represents a 28.8-fold improvement in binding over the best scFv from the directed evolution. Additionally, 99% of the designed scFvs in our most successful library are improvements over the initial candidate scFv. By comparing a librarys predicted success to actual measurements, we demonstrate our methods ability to explore tradeoffs between library success and diversity during the design phase and prior to experimental testing. The results of our work highlight the significant impact machine learning models can have on scFv development. We expect our end-to-end method to be broadly applicable and able to provide value to other protein engineering tasks.

bioinformatics↗

bioRxiv2022

Meiotic Nuclear Pore Complex Remodeling Provides Key Insights into Nuclear Basket Organization

Nuclear pore complexes (NPCs) are large proteinaceous assemblies that mediate nuclear compartmentalization. NPCs undergo largescale structural rearrangements during mitosis in metazoans and some fungi. However, our understanding of NPC remodeling beyond mitosis remains limited. Using time-lapse fluorescence microscopy, we discovered that NPCs undergo two mechanistically-separable remodeling events during budding yeast meiosis whereby parts or all of the nuclear basket transiently dissociate from the NPC core during meiosis I and II, respectively. Meiosis I detachment, observed for Nup60 and Nup2, is driven by Polo kinase-mediated phosphorylation of Nup60 at its interface with the Y-complex. Subsequent reattachment of Nup60-Nup2 to the NPC core is mediated by a lipid-binding amphipathic helix in Nup60. Preventing Nup60-Nup2 reattachment causes misorganization of the entire nuclear basket in gametes. Strikingly, meiotic nuclear basket remodeling also occurs in the distantly related fission yeast, Schizosaccharomyces pombe. Our study reveals a conserved and developmentally programmed aspect of NPC plasticity, providing key mechanistic insights into nuclear basket organization. SUMMARYKing and Wettstein et al. reveal that nuclear pore complexes undergo two distinct remodeling events during budding yeast meiosis: partial and full nuclear basket detachment. By dissecting the regulation of these events, the study provides mechanistic insights into NPC organization.

cell biology↗

bioRxiv2019

Identification of clinically approved small molecules that inhibit growth and promote surface remodeling in the African trypanosome

Trypanosoma brucei are unicellular parasites endemic to Sub-Saharan Africa that cause fatal disease in humans and animals. Infection with these parasites is caused by the bite of the tsetse fly vector, and parasites living extracellularly in the blood of infected animals evade the host immune system through antigenic variation. Existing drugs for Human and Animal African Trypanosomiasis are difficult to administer and can have serious side effects. Resistance to some drugs is also increasing, creating an urgent need for alternative trypanosomiasis therapeutics. In addition to identifying drugs that inhibit trypanosome growth, we wish to identify small molecules that can induce bloodstream form parasites to differentiate into forms adapted for the insect vector. These insect stage parasites do not vary proteins on their cell surface, making them vulnerable to the host immune system. To identify drugs that trigger differentiation of the parasite from bloodstream to insect stages, we engineered bloodstream reporter parasites that express Green Fluorescent Protein (GFP) following induction of the invariant insect-stage specific procyclin transcript. Using these bloodstream reporter strains in combination with high-throughput flow cytometry, we screened a library of 1,585 U.S. or foreign-approved drugs and identified eflornithine, spironolactone, and phenothiazine as small molecules that induce transcription of procylin. Both eflornithine and spironolactone also affect transcript levels for a subset of differentiation associated genes. We further identified 154 compounds that inhibit trypanosome growth. As all of these compounds have already undergone testing for human toxicity, they represent good candidates for repurposing as trypanosome therapeutics. Finally, this study is proof of principle that fluorescent reporters are a useful tool for small molecule or genetic screens aimed at identifying molecules or processes that initiate remodeling of the parasite surface during life cycle stage transitions.\n\nAuthor SummaryAfrican trypanosomes are unicellular parasites that infect humans and animals, causing a fatal disease known as sleeping sickness in humans and nagana in cattle. These diseases impose a severe economic burden for people living in Sub-Saharan Africa, where parasites are transmitted to humans and animals through the bite of the tsetse fly. Parasites living outside cells in humans and animals are attacked by the antibodies of the host immune system, but they can evade this attack by varying the proteins on their cell surface. In contrast, because flies do not have an antibody-mediated immune response, parasites living in flies do not vary the proteins on their cell surface. In this study, we performed a small molecule screen to identify compounds that might force bloodstream parasites to move forward in their life cycle to become more similar to parasites living in flies, causing them to express a protein on their cell surface that does not vary. This invariant protein on the surface of bloodstream parasites would make bloodstream parasites vulnerable to the host antibodies. We found 3 compounds that increased RNA levels for an invariant insect-stage surface protein and 154 compounds that inhibit parasite growth. We hope these compounds might have potential as novel trypanosomiasis therapeutics.

molecular biology↗