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Peptide-based supramolecular assemblies are a promising class of nanomaterials with important biomedical applications, specifically in drug delivery and tissue regeneration. However, the intrinsic antibacterial capabilities of these assemblies have been largely overlooked. The recent identification of common characteristics shared by antibacterial and self-assembling peptides provides a paradigm shift towards development of antibacterial agents. Here we present the antibacterial activity of self-assembled diphenylalanine, which emerges as the minimal model for antibacterial supramolecular polymers. The diphenylalanine nano-assemblies completely inhibit bacterial growth, trigger upregulation of stress-response regulons, induce substantial disruption to bacterial morphology, and cause membrane permeation and depolarization. We demonstrate the specificity of these membrane interactions and the development of antibacterial materials by integration of the peptide assemblies into tissue scaffolds. This study provides important insights into the significance of the interplay between self-assembly and antimicrobial activity and establishes innovative design principles toward the development of antimicrobial agents and materials. Peptide-based supramolecular assemblies are a promising class of nanomaterials with important biomedical applications, but their antibacterial properties can be overlooked. Here the authors show the antibacterial activity of self-assembled diphenylalanine, which emerges as the minimal model for antibacterial supramolecular polymers.

Site-specific protein cleavage is essential for many protein-production protocols and typically requires proteases. We report the development of a chemical protein-cleavage method that is achieved through the use of a sequence-specific nickel-assisted cleavage (SNAC)-tag. We demonstrate that the SNAC-tag can be inserted before both water-soluble and membrane proteins to achieve fusion protein cleavage under biocompatible conditions with efficiency comparable to that of enzymes, and that the method works even when enzymatic cleavages fail.SNAC-tags allow for versatile sequence-specific cleavage of soluble and membrane proteins with Ni2+ under biocompatible conditions, bypassing enzymatic cleavage and enabling cleavage in situations where commonly used enzymes fail.

Transmembrane signaling proteins couple extracytosolic sensors to cytosolic effectors. Here, we examine how binding of Mg 2+ to the sensor domain of an E. coli two component histidine kinase (HK), PhoQ, modulates its cytoplasmic kinase domain. We use cysteine-crosslinking and reporter-gene assays to simultaneously and independently probe the signaling state of PhoQ’s sensor and autokinase domains in a set of over 30 mutants. Strikingly, conservative single-site mutations distant from the sensor or catalytic site strongly influence PhoQ’s ligand-sensitivity as well as the magnitude and direction of the signal. Data from 35 mutants are explained by a semi-empirical three-domain model in which the sensor, intervening HAMP, and catalytic domains can adopt kinase-promoting or inhibiting conformations that are in allosteric communication. The catalytic and sensor domains intrinsically favor a constitutively ‘kinase-on’ conformation, while the HAMP domain favors the ‘off’ state; when coupled, they create a bistable system responsive to physiological concentrations of Mg 2+ . Mutations alter signaling by locally modulating domain intrinsic equilibrium constants and interdomain couplings. Our model suggests signals transmit via interdomain allostery rather than propagation of a single concerted conformational change, explaining the diversity of signaling structural transitions observed in individual HK domains.

Ubiquitin is essential for eukaryotic life and varies in only 3 amino acid positions between yeast and humans. However, recent deep sequencing studies indicate that ubiquitin is highly tolerant to single mutations. We hypothesized that this tolerance would be reduced by chemically induced physiologic perturbations. To test this hypothesis, a class of first year UCSF graduate students employed deep mutational scanning to determine the fitness landscape of all possible single residue mutations in the presence of five different small molecule perturbations. These perturbations uncover 'shared sensitized positions' localized to areas around the hydrophobic patch and the C-terminus. In addition, we identified perturbation specific effects such as a sensitization of His68 in HU and a tolerance to mutation at Lys63 in DTT. Our data show how chemical stresses can reduce buffering effects in the ubiquitin proteasome system. Finally, this study demonstrates the potential of lab-based interdisciplinary graduate curriculum. The ability of an organism to grow and reproduce, that is, it’s “fitness”, is determined by how its genes interact with the environment. Yeast is a model organism in which researchers can control the exact mutations present in the yeast’s genes (its genotype) and the conditions in which the yeast cells live (their environment). This allows researchers to measure how a yeast cell’s genotype and environment affect its fitness. Ubiquitin is a protein that many organisms depend on to manage cell stress by acting as a tag that targets other proteins for degradation. Essential proteins such as ubiquitin often remain unchanged by mutation over long periods of time. As a result, these proteins evolve very slowly. Like all proteins, ubiquitin is built from a chain of amino acid molecules linked together, and the ubiquitin proteins of yeast and humans are made of almost identical sequences of amino acids. Although ubiquitin has barely changed its sequence over evolution, previous studies have shown that – under normal growth conditions in the laboratory – most amino acids in ubiquitin can be mutated without any loss of cell fitness. This led Mavor et al. to hypothesize that treating the yeast cells with chemicals that cause cell stress might lead to amino acids in ubiquitin becoming more sensitive to mutation. To test this idea, a class of graduate students at the University of California, San Francisco grew yeast cells with different ubiquitin mutations together, and with different chemicals that induce cell stress, and measured their growth rates. Sequencing the ubiquitin gene in the thousands of tested yeast cells revealed that three of the chemicals cause a shared set of amino acids in ubiquitin to become more sensitive to mutation. This result suggests that these amino acids are important for the stress response, possibly by altering the ability of yeast cells to target certain proteins for degradation. Conversely, another chemical causes yeast to become more tolerant to changes in the ubiquitin sequence. The experiments also link changes in particular amino acids in ubiquitin to specific stress responses. Mavor et al. show that many of ubquitin’s amino acids are sensitive to mutation under different stress conditions, while others can be mutated to form different amino acids without effecting fitness. By testing the effects of other chemicals, future experiments could further characterize how the yeast’s genotype and environment interact.

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 timescales. 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. Collectively, our experiments have identified eight new sensitizing conditions for Lys63 and 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.

The process by which various upstream sensor and signal-transduction domains of bacterial histidine kinases (HKs) modulate the activity of the conserved autokinase domain remains poorly understood. Specifically, why do most HKs contain modularly inserted signal transduction domains? How do HKs robustly evolve and finetune the coupling between stimulus sensor domains and the conserved autokinase domain, which are often separated by 10s of nanometers? What is the role of these intervening domains in fine-tuning signaling parameters such as the minimum/maximum responsiveness, mid-point, and steepness of signal transition of an HK? In this work, we examine signal transduction through model E. coli HKs, PhoQ and CpxA, which contain one of the most abundant signal transduction domains in HKs, the HAMP domain. We first generate a large set of single-point mutants of PhoQ, and simultaneously measure the signaling state of the ligand-binding sensor and the kinase activity of the autokinase in vitro, at several inducing ligand concentrations to assess the coupling between these two domains. We demonstrate that point mutants in the HAMP signal transduction domain significantly modulate the coupled behavior of the sensor and autokinase, producing markedly varied ligand-dependent responses. We further use the insertion of poly-glycine motifs (Gly7) to decouple domains from one another and qualitatively show that, intrinsically, the sensor domain has a drastically poor ligand-dependent state transition propensity, and similarly, the autokinase domain has a drastically high basal kinase activity. The HAMP domain strongly couples to both domains and is sufficient to adjust these propensities to what is observed in the full length PhoQ. We suggest that signal transduction in PhoQ occurs by an allosteric coupling mechanism, in which the HAMP domain strongly couples to and acts in opposition the underlying signaling state equilibria of PhoQ such that it is maximally responsive to physiologically relevant ranges of stimuli. We demonstrate the same phenomenon in two other E. coli HAMP containing HKs, CpxA and BaeS, and suggest this may be a common theme in the evolution of signal transduction domains in HKs. In order to quantitatively examine the feasibility of modulating various ligand-dependent properties that inform HK function through evolution, we next establish and experimentally fit a three-domain, two-state equilibrium allosteric signaling model. We demonstrate that small changes to the HAMP domain sequence allow for robust modulation of the signaling ensemble and provide quantitative measures for the strong modulation of both sensor and autokinase domains by the HAMP, as well as the effects of point-mutations and Gly7 insertions.We more fully examine the ability of the HAMP to couple strongly and influence the sensor and autokinase domains of PhoQ by introducing a large library of variants in the HAMP four-helix bundle hydrophobic core, as well as the junction between the HAMP and autokinase domains (the S-Helix) and selecting for variants with high PhoQ activity. We find that destabilizing the HAMP four-helix bundle hydrophobic core does indeed lead to higher kinase activity. Furthermore, we find that the wild-type S-Helix sequence is enriched in the high-activity population, along with sequences with comparable polarity or poor helical propensity. Taken together, these observations lend credence to the hypothesis that the thermodynamically preferred signaling state of the HAMP behaves as a negative allosteric regulator of the autokinase, and that this regulation is alleviated by destabilizing the core helical bundle structure as well as the alpha-helical motif that connects it to the autokinase. We investigate this relationship further using a deep learning method to establish sequence-activity predictive relationships and extract structural features that are essential for this behavior. Finally, we examine the question of whether the HAMP domain exists in two distinct structural states, or rather conformational ensembles that can be classified into one of two functional states. We examine signaling through the HAMP domain of an E. coli histidine kinase, CpxA, by constructing a small library of structurally diverse inputs into the HAMP domain and evaluate the resulting autokinase activity as a function of several S-helix point mutations. This analysis allows us to discern the relationship between different signal inputs into the HAMP domain as the linkage to the output domain (autokinase) is varied. We find that the HAMP seems to have a multiconformational landscape that is not explained by 2 unique structural conformations.In this thesis, we show that the insertion of signal transduction domains in HKs can significantly alter both the intrinsic behaviors of sensor and autokinase domains, as well as the coupling between them. These properties can be well-described through a coupled two-state allosteric mechanism, and easily finetuned through simple mutations to the signal transduction domain and its linkage to adjacent domains to achieve the desired physiologically relevant activity profile.

The randomization and screening of combinatorial DNA libraries is a powerful technique for understanding sequence–function relationships and optimizing biosynthetic pathways. Although it can be difficult to predict a priori which sequence combinations encode functional units, it is often possible to omit undesired combinations that inflate library size and screening effort. However, defined library generation is difficult when a complex scan through sequence space is needed. To overcome this challenge, we designed a hybrid valve- and droplet-based microfluidic system that deterministically assembles DNA parts in picoliter droplets, reducing reagent consumption and bias. Using this system, we built a combinatorial library encoding an engineered histidine kinase (HK) based on bacterial CpxA. Our library encodes designed transmembrane (TM) domains that modulate the activity of the cytoplasmic domain of CpxA and variants of the structurally distant “S helix” located near the catalytic domain. We find that the S helix sets a basal activity further modulated by the TM domain. Surprisingly, we also find that a given TM motif can elicit opposing effects on the catalytic activity of different S-helix variants. We conclude that the intervening HAMP domain passively transmits signals and shapes the signaling response depending on subtle changes in neighboring domains. This flexibility engenders a richness in functional outputs as HKs vary in response to changing evolutionary pressures.

Transmembrane signaling proteins couple extracytosolic sensors to cytosolic effectors. Here, we examine how binding of Mg2+ to the sensor domain of an E. coli two component histidine kinase (HK), PhoQ, modulates its cytoplasmic kinase domain. We use cysteine crosslinking and reporter-gene assays to simultaneously and independently probe the signaling state of PhoQ’s sensor and autokinase domains in a set of over 30 mutants. Strikingly, conservative single-site mutants distant from the sensor or catalytic site strongly influence PhoQ’s ligand-sensitivity as well as the magnitude and direction of the signal, endowing diverse signaling characteristics without need for epistasis. Data from 35 mutants are explained by a semi-empirical 3-domain model in which the sensor, intervening HAMP, and catalytic domains can adopt kinase-promoting or inhibiting conformations, that are in allosteric communication. The catalytic and sensor domains intrinsically favor a constitutively ‘kinase-on’ conformation, while the HAMP favors the ‘off’ state; when coupled, they create a bistable system responsive to physiological [Mg2+]. Mutants alter signaling by locally modulating these intrinsic equilibrium constants and couplings. Our model suggests signals transmit via interdomain allostery rather than propagation of a single concerted conformational change, explaining the diversity of signaling structural transitions observed in individual HK domains. Competing Interest Statement The authors have declared no competing interest.

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. Mavor 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, Conchúir SÓ, 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. We 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.

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 reveal novel mutational sensitivities of ubiquitin residues. We found sensitization of Lys63 in eight new conditions. In total, our experiments have uncovered a highly 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.