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Open Access Publications from the University of California

Scholarly Works (12 results)

  • Thesis
  • Peer Reviewed
UC San Francisco Electronic Theses and Dissertations (2019)

Proteins are complex macromolecules whose structure informs their function and regulation in difficult to predict ways. Understanding their shape, dynamics, and regulation all pose major challenges in terms of collecting, analyzing, and interpreting data. In my dissertation I describe two contributions to data analysis for determining the structure and dynamics of proteins using novel approaches, as well as experimental work querying the function of an enzyme with a particularly recalcitrant substrate.

In the first chapter of this dissertation, I develop a tool, EMRinger, for the emerging field of high resolution electron microscopy that takes advantage of prior physical information about model geometry to more effectively determine if the model is built correctly into the map. This work adapted the tool Ringer, which had been previously developed in the Alber lab, in order to identify the dihedral angle for side chains with the greatest density, and confirm that the distribution of those peak positions does not violate the constraints of side chain dihedral angles to rotameric positions. This approach allows for orthogonal validation of backbone position in density (using the side chain density as a “lever”), which generally improves with refinement and is among the most sensitive model-in-map validation tools available for high resolution electron microscopy.

In the second chapter, I present progress on applying temperature jumps to folded proteins to quantify kinetics of the intrinsic motions in proteins that impact their function and regulation. Using a pulsed infrared laser, we raise the temperature of a protein solution in nanoseconds, and follow the progression of the structure of the protein using solution x-ray scattering. As the protein changes temperature, the conformational equilibrium of the protein shifts as higher energy states become more accessible. By following this progress over the nanoseconds, microseconds and milliseconds following the heating pulse, we are able to reconstruct the relaxation of the protein into its new comformational equilibrium. With this information, we can gain kinetic information about the conformational landscapes of our sample, and using mutations we correlate the rates we observe with existing structural models of dynamics that have been characterized by x-ray crystallography.

In the third chapter, I investigate the mechanics of Acidic Mammalian Chitinase, which has the role of breaking down the recalcitrant polysaccharide chitin in the stomach and lungs of mammals. Mutations to acidic mammalian chitinase have previously been identified that lead to either protection against allergic asthma, and previous work has determined that these mutations lead to an increase in the activity of the enzyme. In order to better determine how these mutations affect activity, I developed new methods to assay chitinase activity. I use these methods to characterize the effects of the asthma-associated mutations, as well as investigating the role of the individual domains of acidic mammalian chitinase in degrading crystalline chitin and the differences in behavior of acidic mammalian chitinase and the other chitinase expressed in mammals, chitotriosidase. Additionally, I attempt to engineer hyperactive chitinases, and show that direct evolution based on screening with traditional fluorogenic oligomer substrates does not necessarily lead effectively to enzymes which are hyperactive against complex substrate, emphasizing the need for sensitive and high throughput methods to quantify degradation of crystalline chitin.

    Cover page: Maximizing Interpretability from Complex Experiments in Structural Biology and Biochemistry
    • Article
    • Peer Reviewed
    UC San Francisco Previously Published Works (2015)
    Advances in high-resolution cryo-electron microscopy (cryo-EM) require the development of validation metrics to independently assess map quality and model geometry. We report EMRinger, a tool that assesses the precise fitting of an atomic model into the map during refinement and shows how radiation damage alters scattering from negatively charged amino acids. EMRinger (https://github.com/fraser-lab/EMRinger) will be useful for monitoring progress in resolving and modeling high-resolution features in cryo-EM.
      Cover page: EMRinger: side chain–directed model and map validation for 3D cryo-electron microscopy
      • Article
      • Peer Reviewed
      UC San Francisco Previously Published Works (2020)
      Chitin is an abundant polysaccharide used by many organisms for structural rigidity and water repulsion. As such, the insoluble crystalline structure of chitin poses significant challenges for enzymatic degradation. Acidic mammalian chitinase, a processive glycosyl hydrolase, is the primary enzyme involved in the degradation of environmental chitin in mammalian lungs. Mutations to acidic mammalian chitinase have been associated with asthma, and genetic deletion in mice increases morbidity and mortality with age. We initially set out to reverse this phenotype by engineering hyperactive acidic mammalian chitinase variants. Using a screening approach with commercial fluorogenic substrates, we identified mutations with consistent increases in activity. To determine whether the activity increases observed were consistent with more biologically relevant chitin substrates, we developed new assays to quantify chitinase activity with insoluble chitin, and identified a one-pot fluorogenic assay that is sufficiently sensitive to quantify changes to activity due to the addition or removal of a carbohydrate-binding domain. We show that the activity increases from our directed evolution screen were lost when insoluble substrates were used. In contrast, naturally occurring gain-of-function mutations gave similar results with oligomeric and insoluble substrates. We also show that activity differences between acidic mammalian chitinase and chitotriosidase are reduced with insoluble substrate, suggesting that previously reported activity differences with oligomeric substrates may have been driven by differential substrate specificity. These results highlight the need for assays against physiological substrates when engineering metabolic enzymes, and provide a new one-pot assay that may prove to be broadly applicable to engineering glycosyl hydrolases.
        • Article
        • Peer Reviewed
        UC San Francisco Previously Published Works (2019)
        Correlated motions of proteins are critical to function, but these features are difficult to resolve using traditional structure determination techniques. Time-resolved X-ray methods hold promise for addressing this challenge, but have relied on the exploitation of exotic protein photoactivity, and are therefore not generalizable. Temperature jumps, through thermal excitation of the solvent, have been utilized to study protein dynamics using spectroscopic techniques, but their implementation in X-ray scattering experiments has been limited. Here, we perform temperature-jump small- and wide-angle X-ray scattering measurements on a dynamic enzyme, cyclophilin A, demonstrating that these experiments are able to capture functional intramolecular protein dynamics on the microsecond timescale. We show that cyclophilin A displays rich dynamics following a temperature jump, and use the resulting time-resolved signal to assess the kinetics of conformational changes. Two relaxation processes are resolved: a fast process is related to surface loop motions, and a slower process is related to motions in the core of the protein that are critical for catalytic turnover.
          Cover page: Temperature-jump solution X-ray scattering reveals distinct motions in a dynamic enzyme
          • Article
          • Peer Reviewed
          UC San Francisco Previously Published Works (2019)
          Correlated motions of proteins are critical to function, but these features are difficult to resolve using traditional structure determination techniques. Time-resolved X-ray methods hold promise for addressing this challenge, but have relied on the exploitation of exotic protein photoactivity, and are therefore not generalizable. Temperature jumps, through thermal excitation of the solvent, have been utilized to study protein dynamics using spectroscopic techniques, but their implementation in X-ray scattering experiments has been limited. Here, we perform temperature-jump small- and wide-angle X-ray scattering measurements on a dynamic enzyme, cyclophilin A, demonstrating that these experiments are able to capture functional intramolecular protein dynamics on the microsecond timescale. We show that cyclophilin A displays rich dynamics following a temperature jump, and use the resulting time-resolved signal to assess the kinetics of conformational changes. Two relaxation processes are resolved: a fast process is related to surface loop motions, and a slower process is related to motions in the core of the protein that are critical for catalytic turnover.
            Cover page: Temperature-jump solution X-ray scattering reveals distinct motions in a dynamic enzyme
            • Article
            • Peer Reviewed
            LBL Publications (2018)
              Creative Commons 'BY' version 4.0 license
              • Article
              • Peer Reviewed
              UC San Francisco Previously Published Works (2017)
              The M2 proton channel of influenza A is a drug target that is essential for the reproduction of the flu virus. It is also a model system for the study of selective, unidirectional proton transport across a membrane. Ordered water molecules arranged in "wires" inside the channel pore have been proposed to play a role in both the conduction of protons to the four gating His37 residues and the stabilization of multiple positive charges within the channel. To visualize the solvent in the pore of the channel at room temperature while minimizing the effects of radiation damage, data were collected to a resolution of 1.4 Å using an X-ray free-electron laser (XFEL) at three different pH conditions: pH 5.5, pH 6.5, and pH 8.0. Data were collected on the Inwardopen state, which is an intermediate that accumulates at high protonation of the His37 tetrad. At pH 5.5, a continuous hydrogen-bonded network of water molecules spans the vertical length of the channel, consistent with a Grotthuss mechanism model for proton transport to the His37 tetrad. This ordered solvent at pH 5.5 could act to stabilize the positive charges that build up on the gating His37 tetrad during the proton conduction cycle. The number of ordered pore waters decreases at pH 6.5 and 8.0, where the Inwardopen state is less stable. These studies provide a graphical view of the response of water to a change in charge within a restricted channel environment.
                • Article
                • Peer Reviewed
                UC San Diego Previously Published Works (2024)
                Structural biology efforts using cryogenic electron microscopy are frequently stifled by specimens adopting preferred orientations on grids, leading to anisotropic map resolution and impeding structure determination. Tilting the specimen stage during data collection is a generalizable solution but has historically led to substantial resolution attenuation. Here, we develop updated data collection and image processing workflows and demonstrate, using multiple specimens, that resolution attenuation is negligible or significantly reduced across tilt angles. Reconstructions with and without the stage tilted as high as 60° are virtually indistinguishable. These strategies allowed the reconstruction to 3 Å resolution of a bacterial RNA polymerase with preferred orientation, containing an unnatural nucleotide for studying novel base pair recognition. Furthermore, we present a quantitative framework that allows cryo-EM practitioners to define an optimal tilt angle during data acquisition. These results reinforce the utility of employing stage tilt for data collection and provide quantitative metrics to obtain isotropic maps.
                  Cover page: Overcoming resolution attenuation during tilted cryo-EM data collection.
                  • Article
                  • Peer Reviewed
                  LBL Publications (2023)
                  Understanding and controlling protein motion at atomic resolution is a hallmark challenge for structural biologists and protein engineers because conformational dynamics are essential for complex functions such as enzyme catalysis and allosteric regulation. Time-resolved crystallography offers a window into protein motions, yet without a universal perturbation to initiate conformational changes the method has been limited in scope. Here we couple a solvent-based temperature jump with time-resolved crystallography to visualize structural motions in lysozyme, a dynamic enzyme. We observed widespread atomic vibrations on the nanosecond timescale, which evolve on the submillisecond timescale into localized structural fluctuations that are coupled to the active site. An orthogonal perturbation to the enzyme, inhibitor binding, altered these dynamics by blocking key motions that allow energy to dissipate from vibrations into functional movements linked to the catalytic cycle. Because temperature jump is a universal method for perturbing molecular motion, the method demonstrated here is broadly applicable for studying protein dynamics.
                    Cover page: Mapping protein dynamics at high spatial resolution with temperature-jump X-ray crystallography.
                    • Article
                    UC San Francisco Previously Published Works (2022)

                    Summary

                    Understanding and controlling protein motion at atomic resolution is a hallmark challenge for structural biologists and protein engineers because conformational dynamics are essential for complex functions such as enzyme catalysis and allosteric regulation. Time-resolved crystallography offers a window into protein motions, yet without a universal perturbation to initiate conformational changes the method has been limited in scope. Here we couple a solvent-based temperature jump with time-resolved crystallography to visualize structural motions in lysozyme, a dynamic enzyme. We observed widespread atomic vibrations on the nanosecond timescale, which evolve on the sub-millisecond timescale into localized structural fluctuations that are coupled to the active site. An orthogonal perturbation to the enzyme, inhibitor binding, altered these dynamics by blocking key motions that allow energy to dissipate from vibrations into functional movements linked to the catalytic cycle. Because temperature-jump is a universal method for perturbing molecular motion, the method demonstrated here is broadly applicable for studying protein dynamics.
                      Cover page: Mapping Protein Dynamics at High Spatial Resolution with Temperature-Jump X-ray Crystallography
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