Differences in the chitinolytic activity of mammalian chitinases on soluble and insoluble substrates

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

Mapping protein dynamics at high spatial resolution with temperature-jump X-ray crystallography

温度による酵素の構造変化を分子動画撮影 様々な生体高分子のダイナミクスを決定する新たな方法論. 京都大学プレスリリース. 2023-09-19.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

The twilight of the Liberal Social Contract? On the Reception of Rawlsian Political Liberalism

This chapter discusses the Rawlsian project of public reason, or public justification-based 'political' liberalism, and its reception. After a brief philosophical rather than philological reconstruction of the project, the chapter revolves around a distinction between idealist and realist responses to it. Focusing on political liberalism’s critical reception illuminates an overarching question: was Rawls’s revival of a contractualist approach to liberal legitimacy a fruitful move for liberalism and/or the social contract tradition? The last section contains a largely negative answer to that question. Nonetheless the chapter's conclusion shows that the research programme of political liberalism provided and continues to provide illuminating insights into the limitations of liberal contractualism, especially under conditions of persistent and radical diversity. The programme is, however, less receptive to challenges to do with the relative decline of the power of modern states

Constitutivism

A brief explanation and overview of constitutivism

Theories in Business and Information Systems Engineering

Even though the idea of science enjoys an impressive reputation, there seems to be no precise conception of science. On the one hand, there is no unified definition of the extension of activities subsumed under the notion of science. According to the narrow conception that is common in Anglo-Saxon countries, science is restricted to those disciplines that investigate nature and aim at explanation and prediction of natural phenomena. A wider conception that can be found in various European countries includes social sciences, the humanities and engineering. On the other hand and related to the first aspect, there is still no general consensus on the specific characteristics of scientific discoveries and scientific knowledge

Philosophy of action

The philosophical study of human action begins with Plato and Aristotle. Their influence in late antiquity and the Middle Ages yielded sophisticated theories of action and motivation, notably in the works of Augustine and Aquinas.1 But the ideas that were dominant in 1945 have their roots in the early modern period, when advances in physics and mathematics reshaped philosophy

Cryo-EM model validation recommendations based on outcomes of the 2019 EMDataResource challenge

This paper describes outcomes of the 2019 Cryo-EM Model Challenge. The goals were to (1) assess the quality of models that can be produced from cryogenic electron microscopy (cryo-EM) maps using current modeling software, (2) evaluate reproducibility of modeling results from different software developers and users and (3) compare performance of current metrics used for model evaluation, particularly Fit-to-Map metrics, with focus on near-atomic resolution. Our findings demonstrate the relatively high accuracy and reproducibility of cryo-EM models derived by 13 participating teams from four benchmark maps, including three forming a resolution series (1.8 to 3.1 Å). The results permit specific recommendations to be made about validating near-atomic cryo-EM structures both in the context of individual experiments and structure data archives such as the Protein Data Bank. We recommend the adoption of multiple scoring parameters to provide full and objective annotation and assessment of the model, reflective of the observed cryo-EM map density

Maximizing Interpretability from Complex Experiments in Structural Biology and Biochemistry

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