Biology subjects

Schwarz, D. M. C.

Publications and source records attributed to Schwarz, D. M. C..

3 recordsLinked to original sources

bioRxiv2020

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

Protein conformations are shaped by cellular environments, but how environmental changes alter the conformational landscapes of specific proteins in vivo remains largely uncharacterized, in part due to the challenge of probing protein structures in living cells. Here, we use deep mutational scanning to investigate how a toxic conformation of -synuclein, a dynamic protein linked to Parkinsons disease, responds to perturbations of cellular proteostasis. In the context of a course for graduate students in the UCSF Integrative Program in Quantitative Biology, we screened a comprehensive library of -synuclein missense mutants in yeast cells treated with a variety of small molecules that perturb cellular processes linked to -synuclein biology and pathobiology. We found that the conformation of -synuclein previously shown to drive yeast toxicity--an extended, membrane-bound helix--is largely unaffected by these chemical perturbations, underscoring the importance of this conformational state as a driver of cellular toxicity. On the other hand, the chemical perturbations have a significant effect on the ability of mutations to suppress -synuclein toxicity. Moreover, we find that sequence determinants of -synuclein toxicity are well described by a simple structural model of the membrane-bound helix. This model predicts that -synuclein penetrates the membrane to constant depth across its length but that membrane affinity decreases toward the C terminus, which is consistent with orthogonal biophysical measurements. Finally, we discuss how parallelized chemical genetics experiments can provide a robust framework for inquiry-based graduate coursework.

biochemistry↗

bioRxiv2018

Temperature-Jump Solution X-ray Scattering Reveals Distinct Motions in a Dynamic Enzyme

Correlated motions of proteins and their bound solvent molecules are critical to function, but these features are difficult to resolve using traditional structure determination techniques. Time-resolved methods hold promise for addressing this challenge but have relied on the exploitation of exotic protein photoactivity, and are therefore not generalizable. Temperature-jumps (T-jumps), through thermal excitation of the solvent, have been implemented to study protein dynamics using spectroscopic techniques, but their implementation in X-ray scattering experiments has been limited. Here, we perform T-jump small- and wide-angle X-ray scattering (SAXS/WAXS) measurements on a dynamic enzyme, cyclophilin A (CypA), demonstrating that these experiments are able to capture functional intramolecular protein dynamics. We show that CypA displays rich dynamics following a T-jump, and use the resulting time-resolved signal to assess the kinetics of conformational changes in the enzyme. Two relaxation processes are resolved, which can be characterized by Arrhenius behavior. We also used mutations that have distinct functional effects to disentangle the relationship of the observed relaxation processes. A fast process is related to surface loop motions important for substrate specificity, whereas a slower process is related to motions in the core of the protein that are critical for catalytic turnover. These results demonstrate the power of time-resolved X-ray scattering experiments for characterizing protein and solvent dynamics on the s-ms timescale. We expect the T-jump methodology presented here will be useful for understanding kinetic correlations between local conformational changes of proteins and their bound solvent molecules, which are poorly explained by the results of traditional, static measurements of molecular structure.

biophysics↗

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↗