Biology subjects

Dickinson, M. S.

Publications and source records attributed to Dickinson, M. S..

16 recordsLinked to original sources

bioRxiv2026

Intracellular pathogen targeting by IL32 elicits cell-autonomous immunity

Interferon-{gamma} safeguards humans against intracellular pathogens, yet how most interferon-stimulated genes protect host cells, and how human-adapted pathogens evade these defenses is unclear1,2. Here, we discover a potent immune surveillance and effector circuit executed by an intracellularly acting cytokine, IL32, that targets and restricts phylogenetically distinct vacuolar pathogens, including the bacterium Chlamydia and the microsporidian Encephalitozoon. Quantitative proteomics coupled to a tailored CRISPR screen, uncovered components of the cysteine/Arg N-degron pathway3 that modify IL32 through oxidation-dependent arginylation, thereby enabling the recruitment of the autophagy machinery to pathogen-containing vacuoles. A forward genetics screen in Chlamydia trachomatis, the leading cause of sexually transmitted bacterial infection, identified the secreted virulence factor IncS as an evasion factor that blocks IL32 targeting and shields this human pathogen from xenophagy. These findings establish an IL32-dependent intracellular sensing mechanism linking IFN{gamma} signaling to N-degron-mediated xenophagy, revealing a broadly relevant axis of human host-pathogen conflict.

immunology↗

bioRxiv2025

Molecular mechanism of substrate transport by human peroxisomal ABCD3

Peroxisomes are eukaryotic oxidative organelles involved in numerous metabolic functions that include fatty acid oxidation, bile acid synthesis, and detoxification of reactive oxygen species. ATP-binding cassette transporters of the D subfamily (ABCD1-3) mediate the import of CoA thioesters of fatty acids into the peroxisome. ABCD3, the most abundant of these transporters in the peroxisomal membrane, facilitates the transport of a broad spectrum of substrates including branched-chain fatty acids, very long-chain fatty acids, bile salt intermediates, and dicarboxylic acids. Mutations in ABCD3 are associated with defects in congenital bile acid synthesis and variants of Zellweger syndrome. The structural and functional details of the human ABCD3 transporter remain unclear, despite its significance. In this study, we report the cryogenic sample electron microscopy (cryo-EM) structures of full-length human ABCD3 in its apo state and bound to one of the physiological substrates (phytanoyl-CoA) at resolutions of 3.33 [A] and 3.13 [A], respectively. Our biochemical assays reveal that substrate binding induces ATPase activity in ABCD3, suggesting a substrate-dependent conformational change. Structural comparison of the apo and substrate bound states demonstrate that the substrate interaction brings nucleotide-binding domains closer, providing a mechanistic basis of substrate induced ATPase activity. These findings offer critical insights into the transport mechanism of ABCD3 and lay a structural foundation for understanding its role in peroxisomal metabolite import and related diseases. Significance StatementPeroxisomes are involved in essential cellular metabolic processes that include fatty acid oxidation, bile acid synthesis, and detoxification. The ABCD subfamily of membrane transporter proteins transport fatty acyl-CoA molecules from the cytosol into peroxisomes. Among the ABCD subfamily members, ABCD3 transports branched chain fatty acids and a range of other important metabolites. Dysfunction in ABCD3 has been associated with several inherited and acquired diseases. Using cryo-EM, we determined high-resolution structures of full-length human ABCD3 in both unbound and phytanoyl-CoA-bound forms. We show that substrate binding stimulates ATP hydrolysis activity by apposition of the two nucleotide binding domains of ABCD3, adding structural insight into the mechanism of action of ABCD3 and its role in peroxisomal function and disease.

biochemistry↗

bioRxiv2025

Human giant GTPase GVIN1 forms an antimicrobial coatomer around the intracellular bacterial pathogen Burkholderia thailandensis

Several human pathogens exploit the kinetic forces generated by polymerizing actin to power their intracellular motility. Human cell-autonomous immune responses activated by the cytokine interferon-gamma (IFNg) interfere with such microbial actin-based motility, yet the underlying molecular mechanisms are poorly defined. Here, we identify the IFNg-inducible human giant GTPases GVIN1 as a novel host defense protein that blocks the bacterial pathogen Burkholderia thailandensis from high-jacking the hosts actin polymerization machinery. We found that GVIN1 proteins form a coatomer around cytosolic bacteria and prevent Burkholderia from establishing force-generating actin comet tails. Coatomers formed by a second IFNg-inducible GTPase, human guanylate binding protein 1 (GBP1), constitute a GVIN1-independent but mechanistically related anti-motility pathway. We show that coating with either GVIN1 or GBP1 displaces the Burkholderia outer membrane protein BimA, an actin nucleator that is essential for actin tail formation. Both GVIN1 and GBP1 coatomers require additional IFNg-inducible co-factors to disrupt the membrane localization of BimA, demonstrating the existence of two parallel-acting IFNg-inducible defense modules that evolved to target a virulence trait critical for the pathogenesis of numerous bacterial infectious agents.

immunology↗

bioRxiv2024

Antibodies disrupt bacterial adhesion by ligand mimicry and allosteric interference

A critical step in infections is the attachment of many microorganisms to host cells using lectins that bind surface glycans, making lectins promising antimicrobial targets. Upon binding mannosylated glycans, FimH, the most studied lectin adhesin of type 1 fimbriae in E. coli, undergoes an allosteric transition from an inactive to an active conformation that can act as a catch-bond. Monoclonal antibodies that alter FimH glycan binding in various ways are available, but the mechanisms of these antibodies remain unclear. Here, we use cryoEM, mass spectrometry, binding assays, and molecular dynamics simulations to determine the structure-function relationships underlying antibody-FimH binding. Our study reveals four distinct antibody mechanisms of action: ligand mimicry by an N-linked, high-mannose glycan; stabilization of the ligand pocket in the inactive state; conformational trapping of the active and inactive states; and locking of the ligand pocket through long-range allosteric effects. These structures reveal multiple mechanisms of antibody responses to an allosteric protein and provide blueprints for new antimicrobial that target adhesins.

immunology↗

bioRxiv2024

The Shigella flexneri effector IpaH1.4 facilitates RNF213 degradation and protects cytosolic bacteria against interferon-induced ubiquitylation

A central signal that marshals host defense against many infections is the lymphocyte-derived cytokine interferon-gamma (IFN{gamma}). The IFN{gamma} receptor is expressed on most human cells, and its activation leads to the expression of antimicrobial proteins that execute diverse cell-autonomous immune programs. One such immune program consists of the sequential detection, ubiquitylation, and destruction of intracellular pathogens. Recently, the IFN{gamma}-inducible ubiquitin E3 ligase RNF213 was identified as a pivotal mediator of such a defense axis. RNF213 provides host protection against viral, bacterial, and protozoan pathogens. To establish infections, potentially susceptible intracellular pathogens must have evolved mechanisms that subdue RNF213-controlled cell-autonomous immunity. In support of this hypothesis, we demonstrate here that a causative agent of bacillary dysentery, Shigella flexneri, uses the type III secretion system (T3SS) effector IpaH1.4 to induce the degradation of RNF213. S. flexneri mutants lacking IpaH1.4 expression are bound and ubiquitylated by RNF213 in the cytosol of IFN{gamma}-primed host cells. Linear (M1-) and lysine-linked ubiquitylation of S. flexneri requires RNF213 but is independent of the linear ubiquitin chain assembly complex (LUBAC). We find that ubiquitylation of S. flexneri is insufficient to kill intracellular bacteria, suggesting that S. flexneri employs additional virulence factors to escape from host defenses that operate downstream from RNF213-driven ubiquitylation. In brief, this study identified the bacterial IpaH1.4 protein as an inhibitor of mammalian RNF213 and highlights evasion of RNF213-driven immunity as a characteristic of the human-tropic pathogen Shigella.

microbiology↗

bioRxiv2024

Broadly neutralizing antibodies against emerging delta-coronaviruses

Porcine deltacoronavirus (PDCoV) spillovers were recently detected in children with acute undifferentiated febrile illness, underscoring recurrent zoonoses of divergent coronaviruses. To date, no vaccines or specific therapeutics are approved for use in humans against PDCoV. To prepare for possible future PDCoV epidemics, we isolated human spike (S)-directed monoclonal antibodies from transgenic mice and found that two of them, designated PD33 and PD41, broadly neutralized a panel of PDCoV variants. Cryo-electron microscopy structures of PD33 and PD41 in complex with the PDCoV receptor-binding domain and S ectodomain trimer provide a blueprint of the epitopes recognized by these mAbs, rationalizing their broad inhibitory activity. We show that both mAbs inhibit PDCoV by competitively interfering with host APN binding to the PDCoV receptor-binding loops, explaining the mechanism of viral neutralization. PD33 and PD41 are candidates for clinical advancement, which could be stockpiled to prepare for possible future PDCoV outbreaks.

biochemistry↗

bioRxiv2023

Light-sensitive phosphorylation regulates enzyme activity and filament assembly of human IMPDH1 retinal splice variants

Inosine monophosphate dehydrogenase (IMPDH) is the rate-limiting enzyme in de novo guanosine triphosphate (GTP) synthesis and is controlled by feedback inhibition and allosteric regulation. IMPDH assembles into micron-scale filaments in cells, which desensitizes the enzyme to feedback inhibition by GTP and boosts nucleotide production. The vertebrate retina expresses two tissue-specific splice variants IMPDH1(546) and IMPDH1(595). IMPDH1(546) filaments adopt high and low activity conformations, while IMPDH1(595) filaments maintain high activity. In bovine retinas, residue S477 is preferentially phosphorylated in the dark, but the effects on IMPDH1 activity and regulation are unclear. Here, we generated phosphomimetic mutants to investigate structural and functional consequences of phosphorylation in IMPDH1 variants. The S477D mutation re-sensitized both variants to GTP inhibition, but only blocked assembly of IMPDH1(595) filaments and not IMPDH1(546) filaments. Cryo-EM structures of both variants showed that S477D specifically blocks assembly of the high activity assembly interface, still allowing assembly of low activity IMPDH1(546) filaments. Finally, we discovered that S477D exerts a dominant-negative effect in cells, preventing endogenous IMPDH filament assembly. By modulating the structure and higher-order assembly of IMPDH, phosphorylation at S477 acts as a mechanism for downregulating retinal GTP synthesis in the dark, when nucleotide turnover is decreased. Like IMPDH1, many other metabolic enzymes dynamically assemble filamentous polymers that allosterically regulate activity. Our work suggests that posttranslational modifications may be yet another layer of regulatory control to finely tune activity by modulating filament assembly in response to changing metabolic demands. SIGNIFICANCE STATEMENTOver 20 different metabolic enzymes form micron-scale filaments in cells, suggesting that filament assembly is a conserved mechanism for regulating diverse metabolic pathways. Filament assembly regulates catalytic activity of many of these enzymes, including inosine monophosphate dehydrogenase (IMPDH), the rate-limiting enzyme in de novo GTP biosynthesis. The vertebrate retina expresses two IMPDH1 splice variants that are critical for maintaining nucleotide levels required for phototransduction. Here, we show that filament assembly by these variants is itself controlled by phosphorylation at a single residue, adding further complexity to the tight regulation of nucleotide metabolism in the retina. Phosphorylation and other posttranslational modifications are likely to be a general regulatory mechanism controlling filament assembly by enzymes in many different metabolic pathways.

biochemistry↗

bioRxiv2023

Blueprinting expandable nanomaterials with standardized protein building blocks

A wooden house frame consists of many different lumber pieces, but because of the regularity of these building blocks, the structure can be designed using straightforward geometrical principles. The design of multicomponent protein assemblies in comparison has been much more complex, largely due to the irregular shapes of protein structures1. Here we describe extendable linear, curved, and angled protein building blocks, as well as inter-block interactions that conform to specified geometric standards; assemblies designed using these blocks inherit their extendability and regular interaction surfaces, enabling them to be expanded or contracted by varying the number of modules, and reinforced with secondary struts. Using X-ray crystallography and electron microscopy, we validate nanomaterial designs ranging from simple polygonal and circular oligomers that can be concentrically nested, up to large polyhedral nanocages and unbounded straight "train track" assemblies with reconfigurable sizes and geometries that can be readily blueprinted. Because of the complexity of protein structures and sequence-structure relationships, it has not been previously possible to build up large protein assemblies by deliberate placement of protein backbones onto a blank 3D canvas; the simplicity and geometric regularity of our design platform now enables construction of protein nanomaterials according to "back of an envelope" architectural blueprints.

biochemistry↗

bioRxiv2023

Structure and dynamics of the essential endogenous mycobacterial polyketide synthasePks13

Mycobacterium tuberculosis is currently the leading cause of death by any bacterial infection1. The mycolic acid layer of the cell wall is essential for viability and virulence, and the enzymes responsible for its synthesis are therefore front line targets for antimycobacterial drug development2,3. Polyketide synthase 13 (Pks13) is a module comprised of a closely symmetric parallel dimer of chains, each encoding several enzymatic and transport functions, that carries out the condensation of two different very long chain fatty acids to produce mycolic acids that are essential components of the mycobacterial cell wall. Consequently individual enzymatic domains of Pks13 are targets for antimycobacterial drug development4. To understand this machinery, we sought to determine the structure and domain trajectories of the dimeric multi-enzyme Pks13, a 2x198,426 Dalton complex, from protein purified endogenously from mycobacteria under normal growth conditions, to capture it with normal substrates bound trapped in action. Structures of the multi-domain assembly revealed by cryogenic electron microscopy (cryoEM) define the ketosynthase (KS), linker, and acyltransferase (AT) domains, each at atomic resolution (1.8[A]), with bound substrates defined at 2.4[A] and 2.9[A] resolution. Image classification reveals two distinct structures with alternate locations of the N-terminal acyl carrier protein (termed ACP1a, ACP1b) seen at 3.6[A] and 4.6[A] resolution respectively. These two structures suggest plausible intermediate states, related by a ~60[A] movement of ACP1, on the pathway for substrate delivery from the fatty acyl-ACP ligase (FadD32) to the ketosynthase domain. The linking sequence between ACP1 and the KS includes an 11 amino acid sequence with 6 negatively charged side chains that lies in different positively charged grooves on the KS in ACP1a versus ACP1b structures. This charge complementarity between the extended chain and the grooves suggests some stabilization of these two distinct orientations. Other domains are visible at lower resolution and indicate flexibility relative to the KS-AT core. The chemical structures of three bound endogenous long chain fatty acid substrates with their proximal regions defined in the structures were determined by electrospray ionization mass spectrometry. The domain proximities were probed by chemical cross-linking and identified by mass spectrometry. These were incorporated into integrative structure modeling to define multiple domain configurations that transport the very long fatty acid chains throughout the multistep Pks13 mediated synthetic pathway.

biochemistry↗

bioRxiv2022

GBP2 aggregates LPS and activates the caspase-4 inflammasome independent of the bacterial encapsulation factor GBP1

Sensing and killing of intracellular bacterial pathogens are important features of cell-autonomous immunity. The cytokine gamma-interferon (IFN{gamma}) enhances cell-autonomous immunity through upregulation of interferon stimulated genes such as guanylate binding proteins (GBPs). GBPs promote defense against Gram-negative cytosolic bacteria in part through the induction of an inflammatory cell death pathway called pyroptosis. To activate pyroptosis, GBPs facilitate caspase-4 sensing of the Gram-negative bacterial outer membrane component lipopolysaccharide (LPS). There are seven human GBP paralogs and it is unclear how each GBP contributes to LPS sensing and pyroptosis induction. GBP1 forms a multimeric microcapsule on the surface of cytosolic bacteria through direct interactions with LPS and recruits caspase-4 to bacteria, a process deemed essential for caspase-4 activation. In contrast to GBP1, closely related paralog GBP2 is unable to bind bacteria on its own but requires GBP1 for direct bacterial binding. Unexpectedly, we find that GBP2 overexpression can restore Gram-negative-induced pyroptosis in GBP1KO cells, without GBP2 binding to the bacterial surface. A mutant of GBP1 that lacks the triple arginine motif required for microcapsule formation also rescues pyroptosis in GBP1KO cells, showing that binding to bacteria is dispensable for GBPs to promote pyroptosis. Instead, we find that GBP2, like GBP1, directly binds and aggregates free LPS through protein polymerization. This provides a novel mechanistic framework for non-canonical inflammasome activation where GBP1 or GBP2 assemble cytosol-contaminating LPS into a protein-LPS interface for caspase-4 activation as part of a coordinated host response to Gram-negative bacterial infections. Significance StatementSensing Gram-negative bacterial lipopolysaccharide by human caspase-4 is critical for host defense to intracellular Gram-negative bacterial pathogens. Human guanylate binding proteins (GBPs) facilitate caspase-4 activation in response to Gram-negative infections by a poorly understood mechanism. The prevailing model suggests GBP1 binding to bacteria and consequential recruitment of caspase-4 to the bacterial surface are essential for triggering this host response. Here, we show GBP1 binding to bacteria is dispensable for caspase-4 activation and identify GBP2 as an additional lipopolysaccharide-binding protein that can functionally replace GBP1. We demonstrate that GBP1 and GBP2 share the ability to form lipopolysaccharide-protein complexes, which, we propose, allow caspase-4 activation. Our study provides a new mechanistic framework for cytosolic LPS sensing.

immunology↗

bioRxiv2022

DASH/Dam1 complex mutants stabilize ploidy by weakening kinetochore-microtubule attachments

Forcing budding yeast to chromatinize their DNA with human histones manifests an abrupt fitness cost. We previously proposed chromosomal aneuploidy and missense mutations as two potential modes of adaptation to histone humanization. Here we show that aneuploidy in histone-humanized yeasts is specific to a subset of chromosomes, defined by their centromeric evolutionary origins, however, they are not adaptive. Instead we show that a set of missense mutations in outer kineto-chore proteins drive adaptation to human histones. Further, we characterize the molecular mechanism of two mutants of the outer kinetochore DASH/Dam1 complex, which reduce aneuploidy by suppression of chromosome instability. Molecular modeling and biochemical experiments show that these two mutants likely disrupt a conserved oligomerization interface thereby weakening microtubule attachments. Lastly, we show that one mutant, DAD1E50D, while suppressing chromosome instability in mitosis, leads to gross defects in meiosis. In sum, our data show how a set of point mutations evolved in the histone-humanized yeasts to counterbalance human histone induced chromosomal instability through weakening microtubule interactions, eventually promoting a return to euploidy.

genetics↗

bioRxiv2021

Vicia faba TPC1, a genetically encoded variant of the vacuole Two Pore Channel 1, is hyperexcitable

To fire action-potential-like electrical signals, the vacuole membrane requires the depolarization-activated two-pore channel TPC1, also called Slowly activating Vacuolar SV channel. The TPC1/SV channel, encoded by the TPC1 gene, functions as a voltage-dependent and Ca2+-regulated potassium channel. TPC1 currents are activated by a rise in cytoplasmic Ca2+ but inhibited by luminal Ca2+. In search for species-dependent functional TPC1 channel variants, we studied polymorphic amino acids contributing to luminal Ca2+ sensitivity. We found that the acidic residues E457, E605 and D606 of the Ca2+-sensitive Arabidopsis AtTPC1 channel were neutralized by either asparagine or alanine in Vicia faba and many other Fabaceae as well. When expressed in the Arabidopsis loss-of-AtTPC1 function background, the wild type VfTPC1 was hypersensitive to vacuole depolarization and insensitive to blocking luminal Ca2+. When AtTPC1 was mutated for the three VfTPC1-homologous polymorphic site residues, the Arabidopsis At-VfTPC1 channel mutant gained VfTPC1-like voltage and luminal Ca2+ insensitivity that together made vacuoles hyperexcitable. These findings indicate that natural TPC1 channel variants in plant families exist which differ in vacuole excitability and very likely respond to changes in environmental settings of their ecological niche. Significance statementVacuolar electrical excitability and stress-related Ca2+ signaling depends on the activity of the vacuolar cation channel TPC1. Until now, the regulatory features of AtTPC1 from the model plant Arabidopsis thaliana was believed to apply to the TPC1 channels of other species. However, here we now show that, surprisingly, the VfTPC1 channel of the economic broad bean, in contrast to AtTPC1, proves to be hyperactive and confers hyperexcitability to the vacuole. The different gating behavior is most likely related to an impaired Ca2+ sensor site in the vacuolar pore vestibule, rising the probability to open at more negative membrane voltages. These natural variants of the TPC1 channel could help the plant adapt and respond to environmental challenges.

plant biology↗

bioRxiv2021

Symmetry reduction in a hyperpolarization-activated homotetrameric ion channel

Plants obtain nutrients from the soil via transmembrane transporters and channels in their root hairs, from which ions radially transport in towards the xylem for distribution across the plant body. We determined structures of the hyperpolarization-activated channel AKT1 from Arabidopsis thaliana, which mediates K+ uptake from the soil into plant roots. These structures of AtAKT1 embedded in lipid nanodiscs show that the channel undergoes a reduction of C4 to C2 symmetry, possibly to regulate its electrical activation.

biophysics↗

bioRxiv2021

CryoEM and AI reveal a structure of SARS-CoV-2 Nsp2, a multifunctional protein involved in key host processes.

The SARS-CoV-2 protein Nsp2 has been implicated in a wide range of viral processes, but its exact functions, and the structural basis of those functions, remain unknown. Here, we report an atomic model for full-length Nsp2 obtained by combining cryo-electron microscopy with deep learning-based structure prediction from AlphaFold2. The resulting structure reveals a highly-conserved zinc ion-binding site, suggesting a role for Nsp2 in RNA binding. Mapping emerging mutations from variants of SARS-CoV-2 on the resulting structure shows potential host-Nsp2 interaction regions. Using structural analysis together with affinity tagged purification mass spectrometry experiments, we identify Nsp2 mutants that are unable to interact with the actin-nucleation-promoting WASH protein complex or with GIGYF2, an inhibitor of translation initiation and modulator of ribosome-associated quality control. Our work suggests a potential role of Nsp2 in linking viral transcription within the viral replication-transcription complexes (RTC) to the translation initiation of the viral message. Collectively, the structure reported here, combined with mutant interaction mapping, provides a foundation for functional studies of this evolutionary conserved coronavirus protein and may assist future drug design.

biophysics↗

bioRxiv2018

Proximity-dependent proteomics of the Chlamydia trachomatis inclusion membrane reveals functional interactions with endoplasmic reticulum exit sites

Chlamydia trachomatis is the most common bacterial sexually transmitted infection, responsible for millions of infections each year. Despite this high prevalence, the elucidation of the molecular mechanisms of Chlamydia pathogenesis has been difficult due to limitations in genetic tools and its intracellular developmental cycle. Within a host epithelial cell, chlamydiae replicate within a vacuole called the inclusion. Many Chlamydia-host interactions are thought to be mediated by the Inc family of type III secreted proteins that are anchored in the inclusion membrane, but their array of host targets are largely unknown. To investigate how the inclusion membrane proteome changes over the course of an infected cell, we have adapted the APEX system of proximity-dependent biotinylation. APEX is capable of specifically labeling proteins within a 20 nm radius in living cells. We transformed C. trachomatis to express the enzyme APEX fused to known inclusion membrane proteins, allowing biotinylation and pull-down of inclusion-associated proteins. Using quantitative mass spectrometry against APEX labeled samples, we identified over 400 proteins associated with the inclusion membrane at early, middle, and late stages of epithelial cell infection. This system was sensitive enough to detect inclusion interacting proteins early in the developmental cycle, at 8 hours post infection, a previously intractable time point. Mass spectrometry analysis revealed a novel, early association between C. trachomatis inclusions and endoplasmic reticulum exit sites (ERES), functional regions of the ER where COPII-coated vesicles originate. Pharmacological and genetic disruption of ERES function severely restricted early chlamydial growth and the development of infectious progeny. APEX is therefore a powerful in situ approach for identifying critical protein interactions on the membranes of pathogen-containing vacuoles. Furthermore, the data derived from proteomic mapping of Chlamydia inclusions has illuminated an important functional role for ERES in promoting chlamydial developmental growth.

microbiology↗

bioRxiv2017

Extending Chemical Perturbations Of The Ubiquitin Fitness Landscape In A Classroom Setting

Although the primary protein sequence of ubiquitin (Ub) is extremely stable over evolutionary time, it is highly tolerant to mutation during selection experiments performed in the laboratory. We have proposed that this discrepancy results from the difference between fitness under laboratory culture conditions and the selective pressures in changing environments over evolutionary time scales. Building on our previous work (Mavor et al 2016), we used deep mutational scanning to determine how twelve new chemicals (3-Amino-1,2,4-triazole, 5-fluorocytosine, Amphotericin B, CaCl2, Cerulenin, Cobalt Acetate, Menadione, Nickel Chloride, p-fluorophenylalanine, Rapamycin, Tamoxifen, and Tunicamycin) reveal novel mutational sensitivities of ubiquitin residues. We found sensitization of Lys63 in eight new conditions. In total, our experiments have uncovered a sensitizing condition for every position in Ub except Ser57 and Gln62. By determining the Ubiquitin fitness landscape under different chemical constraints, our work helps to resolve the inconsistencies between deep mutational scanning experiments and sequence conservation over evolutionary timescales.\n\nBuilds onMavor D, Barlow KA, Thompson S, Barad BA, Bonny AR, Cario CL, Gaskins G, Liu Z, Deming L, Axen SD, Caceres E, Chen W, Cuesta A, Gate R, Green EM, Hulce KR, Ji W, Kenner LR, Mensa B, Morinishi LS, Moss SM, Mravic M, Muir RK, Niekamp S, Nnadi CI, Palovcak E, Poss EM, Ross TD, Salcedo E, See S, Subramaniam M, Wong AW, Li J, Thorn KS, Conchuir SO, Roscoe BP, Chow ED, DeRisi JL, Kortemme T, Bolon DN, Fraser JS. Determination of Ubiquitin Fitness Landscapes Under Different Chemical Stresses in a Classroom Setting. eLife. 2016.\n\nImpact StatementWe organized a project-based course that used deep mutational scanning in multiple chemical conditions to resolve the inconsistencies between tolerance to mutations in laboratory conditions and sequence conservation over evolutionary timescales.

bioinformatics↗