Browsing by keyword "fitness landscape"
Now showing items 1-2 of 2
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Constrained mutational sampling of amino acids in HIV-1 protease evolutionThe evolution of HIV-1 protein sequences should be governed by a combination of factors including nucleotide mutational probabilities, the genetic code, and fitness. The impact of these factors on protein sequence evolution are interdependent, making it challenging to infer the individual contribution of each factor from phylogenetic analyses alone. We investigated the protein sequence evolution of HIV-1 by determining an experimental fitness landscape of all individual amino acid changes in protease. We compared our experimental results to the frequency of protease variants in a publicly available dataset of 32,163 sequenced isolates from drug-naive individuals. The most common amino acids in sequenced isolates supported robust experimental fitness, indicating that the experimental fitness landscape captured key features of selection acting on protease during viral infections of hosts. Amino acid changes requiring multiple mutations from the likely ancestor were slightly less likely to support robust experimental fitness than single mutations, consistent with the genetic code favoring chemically conservative amino acid changes. Amino acids that were common in sequenced isolates were predominantly accessible by single mutations from the likely protease ancestor. Multiple mutations commonly observed in isolates were accessible by mutational walks with highly fit single mutation intermediates. Our results indicate that the prevalence of multiple base mutations in HIV-1 protease is strongly influenced by mutational sampling.
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Determination of Ubiquitin Fitness Landscapes Under Different Chemical Stresses in a Classroom Setting [preprint]Ubiquitination is an essential post-translational regulatory process that can control protein stability, localization, and activity. Ubiquitin is essential for eukaryotic life and is highly conserved, varying in only 3 amino acid positions between yeast and humans. However, recent deep sequencing studies in S. cerevisiae indicate that ubiquitin is highly tolerant to single amino acid mutations. To resolve this paradox, we hypothesized that the set of tolerated substitutions would be reduced when the cultures are not grown in rich media conditions and that chemically induced physiologic perturbations might unmask constraints on the ubiquitin sequence. To test this hypothesis, a class of first year UCSF graduate students employed a deep mutational scanning procedure to determine the fitness landscape of a library of all possible single amino acid mutations of ubiquitin in the presence of one of five small molecule perturbations: MG132, Dithiothreitol (DTT), Hydroxyurea (HU), Caffeine, and DMSO. Our data reveal that the number of tolerated substitutions is greatly reduced by DTT, HU, or Caffeine, and that these perturbations uncover “shared sensitized positions” localized to areas around the hydrophobic patch and to the C-terminus. We also show perturbation specific effects including the sensitization of His68 in HU and tolerance to mutation at Lys63 in DTT. Taken together, our data suggest that chemical stress reduces buffering effects in the ubiquitin proteasome system, revealing previously hidden fitness defects. By expanding the set of chemical perturbations assayed, potentially by other classroom-based experiences, we will be able to further address the apparent dichotomy between the extreme sequence conservation and the experimentally observed mutational tolerance of ubiquitin. Finally, this study demonstrates the realized potential of a project lab-based interdisciplinary graduate curriculum.
