Journal: Frontiers in Bioengineering and Biotechnology

Article Title: Evaluation of Heterologous Biosynthetic Pathways for Methanol-Based 5-Aminovalerate Production by Thermophilic Bacillus methanolicus

doi: 10.3389/fbioe.2021.686319

Figure Lengend Snippet: Bacterial strains, plasmids, and primers used in this study.

Article Snippet: The L -lysine-α-oxidase-coding regions from Trichoderma viride (GenBank AB937978.1) and S. japonicus (GenBank AB970726.1) were codon-optimized for B. methanolicus MGA3 expression and synthesized by Twist Biosciences ( and ).

Techniques: Cloning, Mutagenesis, Plasmid Preparation, Gene Expression, Control, Low Copy Number, Expressing, Derivative Assay, Sequencing

Journal: bioRxiv

Article Title: Robust Sequence Determinants of α-Synuclein Toxicity in Yeast Implicate Membrane Binding

doi: 10.1101/2020.05.01.072884

Figure Lengend Snippet: Previously mapped sequence–toxicity landscape of α-synuclein identifies a toxic molecular conformation. (A) Fitness scores (defined as the slope of the line describing change in log-transformed variant frequencies over time, represented by a red-white-blue color scale) for expression of α-synuclein missense mutants in yeast. (B) Average fitness scores of mutants with hydrophobic (W, Y, F, L, I, V, M, C, A; red), polar (S, T, N, Q, H, R, K, D, E; blue), or proline (green) residues. (C) The structural model derived from the sequence–toxicity landscape in panel A: an extended, membrane-bound 11/3-helix with increasing dynamics toward the C terminus. These results were obtained previously and are revisited here to provide context for the effects of cellular perturbations on the fitness landscape.

Article Snippet: A pooled double-stranded DNA library containing the sequences of 2,660 α-synuclein mutants was generously provided by Twist Bioscience and cloned into pYES2-αSyn-EGFP, which was generously provided by the laboratory of V. M.-Y.

Techniques: Sequencing, Transformation Assay, Variant Assay, Expressing, Derivative Assay

Journal: bioRxiv

Article Title: Robust Sequence Determinants of α-Synuclein Toxicity in Yeast Implicate Membrane Binding

doi: 10.1101/2020.05.01.072884

Figure Lengend Snippet: Chemical genetics approach to probe sequence and structural determinants of α-synuclein toxicity. A pooled library of yeast cells, each expressing a different missense variant of α-synuclein (represented by yeast cells with differently colored outlines), is treated with a variety of small molecules to perturb cellular proteostasis (represented by different intracellular shading [i.e., white vs. grey]). Induction of α-synuclein expression creates a selective pressure that causes changes in the relative frequency of each missense variant in the population. This change in frequency can be quantified by counting the number of occurrences of each variant in the population over time using deep sequencing. The resulting sequence–toxicity landscape, where the relative toxicity of each variant is represented by a red-white-blue color scale, reveals structural features that can implicate specific conformational states as drivers of toxicity.

Article Snippet: A pooled double-stranded DNA library containing the sequences of 2,660 α-synuclein mutants was generously provided by Twist Bioscience and cloned into pYES2-αSyn-EGFP, which was generously provided by the laboratory of V. M.-Y.

Techniques: Sequencing, Expressing, Variant Assay

Journal: bioRxiv

Article Title: Robust Sequence Determinants of α-Synuclein Toxicity in Yeast Implicate Membrane Binding

doi: 10.1101/2020.05.01.072884

Figure Lengend Snippet: Conformational signatures of toxic α-synuclein species in yeast are robust to diverse chemical perturbations. (A) Average fitness scores of mutants with polar (S, T, N, Q, H, R, K, D, E) or proline residues in untreated cells. These data were collected previously and are shown here for comparison. (B) Average fitness scores of mutants with polar residues when assayed in yeast cells perturbed by small molecules. Each column reflects the average fitness scores of mutants with polar substitutions at a given position. Each row reflects the fitness scores for a different treatment. Fitness scores are normalized for comparison and shown as a red-white-blue color scale. Across all conditions, α-helical periodicity in the mutational signature is retained.

Article Snippet: A pooled double-stranded DNA library containing the sequences of 2,660 α-synuclein mutants was generously provided by Twist Bioscience and cloned into pYES2-αSyn-EGFP, which was generously provided by the laboratory of V. M.-Y.

Techniques:

Journal: bioRxiv

Article Title: Robust Sequence Determinants of α-Synuclein Toxicity in Yeast Implicate Membrane Binding

doi: 10.1101/2020.05.01.072884

Figure Lengend Snippet: Fitness scores of yeast expressing α-synuclein missense variants obtained in the presence of the compounds in , relative to those obtained in untreated controls.

Article Snippet: A pooled double-stranded DNA library containing the sequences of 2,660 α-synuclein mutants was generously provided by Twist Bioscience and cloned into pYES2-αSyn-EGFP, which was generously provided by the laboratory of V. M.-Y.

Techniques: Expressing

Journal: bioRxiv

Article Title: Robust Sequence Determinants of α-Synuclein Toxicity in Yeast Implicate Membrane Binding

doi: 10.1101/2020.05.01.072884

Figure Lengend Snippet: (A) Fitness scores of cells expressing α-synuclein mutants [labeled as (position, amino-acid substitution)] obtained in the presence of rapamycin relative to untreated controls. (B) Helical wheel of α-synuclein depicting the relative orientation of each residue in the 11-residue repeat. Schematic coloring reflects the physicochemical properties of the moiety/environment (blue: positively charged; red: negatively charged; purple: polar; grey: nonpolar). (C) Volcano plot describing changes in fitness score induced by rapamycin treatment, averaged over equivalent mutations in each of the seven repeating 11-residue segments, labeled as (position, amino-acid substitution). The significance of these changes was then determined from their z-scores, assuming a normal distribution, yielding the p-values shown. A 5% false discovery rate threshold is shown, which identifies the significance value above which 5% of the values would be falsely predicted as significantly different in the presence of compound; this threshold was determined by Benjamini-Hochberg method.

Article Snippet: A pooled double-stranded DNA library containing the sequences of 2,660 α-synuclein mutants was generously provided by Twist Bioscience and cloned into pYES2-αSyn-EGFP, which was generously provided by the laboratory of V. M.-Y.

Techniques: Expressing, Labeling

Journal: bioRxiv

Article Title: Robust Sequence Determinants of α-Synuclein Toxicity in Yeast Implicate Membrane Binding

doi: 10.1101/2020.05.01.072884

Figure Lengend Snippet: Cellular toxicity (represented by a red-white-blue color scale) depends on the membrane-bound population of α-synuclein. (A) In untreated cells, WT α-synuclein binds to cellular membranes above a critical threshold for toxicity. Mutations can reduce toxicity by decreasing the membrane-bound population of α-synuclein, either by decreasing membrane affinity (e.g., by introducing polar amino acids on the membrane-binding face) or reducing α-synuclein expression (e.g., by substituting Met-1). (B) Chemical stresses increase sensitivity to α-synuclein toxicity, so mutations that reduce toxicity in untreated cells are less effective at reducing toxicity under general chemical stress. (C) Because rapamycin reduces protein expression (black arrows relative to gray arrows), the combination of rapamycin and specific α-synuclein mutations reduces membrane binding sufficiently to overcome the increased sensitivity caused by compound toxicity.

Article Snippet: A pooled double-stranded DNA library containing the sequences of 2,660 α-synuclein mutants was generously provided by Twist Bioscience and cloned into pYES2-αSyn-EGFP, which was generously provided by the laboratory of V. M.-Y.

Techniques: Binding Assay, Expressing

Journal: bioRxiv

Article Title: Robust Sequence Determinants of α-Synuclein Toxicity in Yeast Implicate Membrane Binding

doi: 10.1101/2020.05.01.072884

Figure Lengend Snippet: A thermodynamic model for α-synuclein–membrane interactions reveals determinants of decreasing mutational sensitivity. (A) The depth of insertion (green) and transfer free energy (blue) of each repeated 11-residue membrane-binding segment of α-synuclein were estimated by optimizing correlation between experimental toxicity scores and changes in the membrane-bound population of α-synuclein predicted based on the energy required to transfer an amphiphilic helix with the appropriate sequence to a particular depth within the lipid bilayer. See text for additional details. (B) Predicted changes in membrane-bound α-synuclein caused by missense mutations, based on the optimal penetration depth and transfer free energy of the WT protein determined in panel A and the depth-dependent transfer free energy of each amino acid. See text for additional details. Relative change in fraction bound is denoted as a red-white-blue color scale. (C) Observed changes in yeast toxicity (represented by a red-white-blue color scale) caused by α-synuclein missense mutations. The Pearson R 2 correlation coefficient between all predicted and observed scores is 0.57.

Article Snippet: A pooled double-stranded DNA library containing the sequences of 2,660 α-synuclein mutants was generously provided by Twist Bioscience and cloned into pYES2-αSyn-EGFP, which was generously provided by the laboratory of V. M.-Y.

Techniques: Binding Assay, Sequencing