Biology subjects

Thorne, R. E.

Publications and source records attributed to Thorne, R. E..

5 recordsLinked to original sources

bioRxiv2025

Activation parameters, enthalpy-entropy compensation and the temperature-dependent activity of enzymes

The increase in enzyme-catalyzed reaction rates with temperature is typically modeled using Arrhenius or Eyring relations. Interpretation of extracted parameters is subject to multiple caveats. Here we analyze the impact of expected small temperature variations of underlying activation/Eyring parameters on this modeling. Linear Arrhenius/Eyring behavior can still be observed when the underlying activation energy Ea or enthalpy {Delta}H and entropy {Delta}S vary with temperature. Modest variations -- of the order of an H-bond energy over 60 {degrees}C -- lead to large fractional deviations of Ea, {Delta}H and {Delta}S values derived from linear fits from their underlying values and to deviations of Arrhenius prefactors A by orders of magnitude. In a family of related enzymes with similar activation free energies {Delta}G, small differences in temperature variation of {Delta}H and {Delta}S will lead to apparent enthalpy-entropy compensation and may scramble enzyme ordering based on {Delta}H or {Delta}S. For enzymes from cold and warm-adapted species having largely similar active sites, small temperature variations of {Delta}H and {Delta}S may explain large differences in apparent {Delta}H values. Similar considerations apply to interpretation of van t Hoff plots of equilibrium measurements and related observations of enthalpy-entropy compensation. Complementary methods including simulations and multi-temperature static and time-resolved atomic-resolution structural studies should play a key role in interpreting temperature-dependent kinetic and equilibrium data from enzymatic systems. One-Sentence SummaryLinear Arrhenius/Eyring behavior can occur even when enzyme structure and interactions vary with temperature, yielding fit parameters far from underlying values and generating apparent enthalpy-entropy compensation.

biophysics↗

bioRxiv2024

A structural perspective on the temperature-dependent activity of enzymes

Enzymes are biomolecular catalysts whose activity varies with temperature. Unlike for small-molecule catalysts, the structural ensembles of enzymes can vary substantially with temperature, and it is in general unclear how this modulates the temperature dependence of activity. Here multi-temperature X-ray crystallography was used to record structural changes from -20{degrees}C to 40{degrees}C for a mesophilic enzyme in complex with inhibitors mimicking substrate-, intermediate-, and product-bound states, representative of major complexes underlying the kinetic constant kcat. Both inhibitors, substrates and catalytically relevant loop motifs increasingly populate catalytically competent conformations as temperature increases. These changes occur even in temperature ranges where kinetic measurements show roughly linear Arrhenius/Eyring behavior where parameters characterizing the system are assumed to be temperature independent. Simple analysis shows that linear Arrhenius/Eyring behavior can still be observed when the underlying activation energy / enthalpy values vary with temperature, e.g., due to structural changes, and that the underlying thermodynamic parameters can be far from values derived from Arrhenius/Eyring model fits. Our results indicate a critical role for temperature-dependent atomic-resolution structural data in interpreting temperature-dependent kinetic data from enzymatic systems. One-Sentence SummaryStructural data spanning a 60{degrees}C temperature range for enzyme complexes mimicking the substrate-, intermediate-, and product-bound states illuminate how small temperature-dependent structural changes may modulate activity and render parameters deduced from Arrhenius/Eyring plots unreliable.

biophysics↗

bioRxiv2023

Ice formation and its elimination in cryopreservation of bovine oocytes

Damage from ice and potential toxicity of ice-inhibiting cryoprotective agents (CPAs) are key issues in assisted reproduction using cryopreserved oocytes and embryos. We use synchrotron-based time-resolved x-ray diffraction and tools from protein cryocrystallography to characterize ice formation within bovine oocytes after cooling at rates between [~]1000 {degrees}C/min and [~]600,000{degrees}C /min and during warming at rates between 20,000 and 150,000 {degrees}C /min. Maximum crystalline ice diffraction intensity, maximum ice volume, and maximum ice grain size are always observed during warming. All decrease with increasing CPA concentration, consistent with the decreasing free water fraction. With the cooling rates, warming rates and CPA concentrations of current practice, oocytes may show no ice after cooling but always develop substantial ice fractions on warming, and modestly reducing CPA concentrations causes substantial ice to form during cooling. With much larger cooling and warming rates achieved using cryocrystallography tools, oocytes soaked as in current practice remain essentially ice free during both cooling and warming, and when soaked in half-strength CPA solution oocytes remain ice free after cooling and develop small grain ice during warming. These results clarify the roles of cooling, warming, and CPA concentration in generating ice in oocytes, establish the character of ice formed, and suggest that substantial further improvements in warming rates are feasible. Ice formation can be eliminated as a factor affecting post-thaw oocyte viability and development, allowing other deleterious effects of the cryopreservation cycle to be studied, and osmotic stress and CPA toxicity reduced. Significance StatementCryopreservation of oocytes and embryos is critical in assisted reproduction of humans and domestic animals and in preservation of endangered species. Success rates are limited by damage from crystalline ice, toxicity of cryoprotective agents (CPAs), and damage from osmotic stress. Time-resolved x-ray diffraction of bovine oocytes shows that ice forms much more readily during warming than during cooling, that maximum ice fractions always occur during warming, and that the tools and large CPA concentrations of current protocols can at best only prevent ice formation during cooling. Using tools from cryocrystallography that give dramatically larger cooling and warming rates, ice formation can be completely eliminated and required CPA concentrations substantially reduced, expanding the scope for species-specific optimization of post-thaw reproductive outcomes.

biophysics↗

bioRxiv2021

Millisecond Mix-and-Quench Crystallography (MMQX) Enables Time-Resolved Studies of PEPCK With Remote Data Collection

Time-resolved crystallography of biomolecules in action has advanced rapidly as methods for serial crystallography have improved, but the large number of crystals and complex experimental infrastructure required remain serious obstacles to widespread application. We have developed Millisecond Mix-and-Quench crystallography (MMQX), which yields millisecond time-resolved data using far fewer crystals and routine remote synchrotron data collection. To demonstrate the capabilities of MMQX, the conversion of oxaloacetic acid to phosphoenolpyruvate by phosphoenolpyruvate carboxykinase is observed with a time resolution of 40 ms. MMQX, by lowering the entry barrier to time-resolved crystallography, should enable broad expansion in structural studies of protein dynamics.

biochemistry↗

bioRxiv2019

A Mechanism for Beam-Induced Motion in Cryo-Electron Microscopy

Estimates of heat transfer rates during plunge cooling and patterns of ice observed in cryo-EM samples indicate that the grid bars cool much more slowly than do the support foil and sample near the middle of the grid openings. The resulting transient temperature differences generate transient tensile stresses in the support foil. Most of this foil stress develops while the sample is liquid and cooling toward its glass transition Tg, and so does not generate tensile sample stress. As the grid bars continue cooling toward the cryogen temperature and contracting, the tensile stress in the foil is released, placing the sample in compressive stress. Radiation-induced creep in the presence of this compressive stress should generate a doming of the sample in the foil openings, as is observed experimentally. Crude estimates of the magnitude of doming that may be generated by this mechanism are consistent with observation. Several approaches to reducing beam-induced motion are discussed.

biophysics↗