Biology subjects

Seelig, A.

Publications and source records attributed to Seelig, A..

6 recordsLinked to original sources

bioRxiv2024

Pressure protein denaturation compared to thermal and chemical unfolding - Analyses with cooperative models

The thermodynamics of pressure induced protein unfolding (denaturation) could so far not be directly compared with protein unfolding induced by temperature or chemical agents. Here we provide a new cooperative model for pressure induced protein denaturation, that allows the quantitative comparison of the three denaturing processes based on their free energy, enthalpy, entropy, and cooperativity. As model proteins we use apolipoprotein A-1 and lysozyme. The comparison shows that heat-induced unfolding is the most cooperative process. It is characterized by large positive enthalpies and entropies and (due to enthalpy-entropy compensation) a small negative free energies. Pressure denaturation is less cooperative. The entropies and enthalpies are less positive, and the resulting free energies are more negative. Chemically induced unfolding is least cooperative and shows the most negative free energies, in particular, if guanidinium hydrochloride (exhibiting a high binding affinity to certain proteins) is used as a denaturant. The three unfolding processes not only differ with respect to their cooperativity and the thermodynamic parameters, but also with respect to the volume changes. Whereas thermal and chemical denaturation increase the protein volume, denaturation by pressure reduces the protein volume, suggesting significant structural differences of the denatured proteins. Using cooperative models for protein analyses thus yields significant new insights into the protein unfolding/folding processes.

biophysics↗

bioRxiv2023

Chemical protein unfolding - A simple cooperative model

Chemical unfolding with guanidineHCl or urea is a common method to study the conformational stability of proteins. The analysis of unfolding isotherms is usually performed with an empirical linear extrapolation method (LEM). The method contradicts however, common expectations. A large positive free energy is assigned to the native protein which is usually considered to be a minimum of the free energy. Here we present a multistate cooperative model, which addresses specifically the binding of the denaturant to the protein and the cooperativity of the protein unfolding equilibrium. The model is based on a molecular statistical-mechanical partition function of the ensemble but simple solutions for the calculation of the binding isotherm and the associated free energy are presented. The model is applied to 23 published unfolding isotherms of small and large proteins. For a given denaturant, the binding constant depends on temperature and pH, but shows little protein specificity. Chemical unfolding is less cooperative than thermal unfolding. The cooperativity parameter {sigma} is two orders of magnitude larger than that of thermal unfolding. The multistate cooperative model predicts a zero free energy for the native protein, which becomes strongly negative beyond the midpoint concentration of unfolding. The free energy to unfold a cooperative unit corresponds exactly to the diffusive energy of the denaturant concentration gradient necessary for unfolding. The temperature dependence of unfolding isotherms yields the denaturant-induced unfolding entropy and, in turn, the unfolding enthalpy. The multistate cooperative model provides molecular insight, is as simple to apply as the LEM, but avoids the conceptual difficulties of the latter.

biophysics↗

bioRxiv2022

Protein unfolding. Thermodynamic perspectives and unfolding models.

Protein unfolding is a dynamic cooperative process with many short-lived intermediates. Cooperativity means that similar molecular elements act dependently on each other. The thermodynamics of protein unfolding can be determined with differential scanning calorimetry (DSC). The measurement of the heat capacity provides the temperature profiles of enthalpy, entropy and free energy. The thermodynamics of protein unfolding is completely determined with these thermodynamic properties. We emphasise the model-independent analysis of the heat capacity. The temperature profiles of enthalpy H(T), entropy S(T) and free energy G(T) can be obtained directly by a numerical integration of Cp(T). In evaluating different models for protein unfolding. It is essential to simulate all thermodynamic properties, not only the heat capacity. A chemical equilibrium two-state model is a widely used approximation to protein unfolding. The model assumes a chemical equilibrium between only two protein conformations, the native protein (N) and the unfolded protein (U). The model fits the heat capacity Cp(T) quite well, but fails in simulating the other thermodynamic properties. In this review we propose a modification of the chemical equilibrium two-state model, which removes these inconsistencies. We also propose a new statistical-mechanical two-state model based on a simple, two-parameter partition function Z(T), from which all thermodynamic parameters can be derived. The thermodynamic predictions of the new models are compared to published DSC-experiments obtained with lysozyme, a globular protein, and {beta}-lactoglobulin, a {beta}-barrel protein. Good fits to all thermodynamic properties are obtained. In particular, the models predict a zero free energy for the native protein, which is confirmed experimentally by DSC. This is in contrast to the often-cited chemical equilibrium two-state model, which predict a positive free energy for the native protein. Two-state models use macroscopic fit parameters, the conformational enthalpy and the heat capacity difference between native and unfolded protein. These simulations provide no molecular insight. The review therefore includes a recently published multistate cooperative model based on physicality well-defined molecular parameters only.

biophysics↗

bioRxiv2021

Molecular Understanding of Calorimetric Protein Unfolding Experiments

Protein unfolding is a dynamic cooperative equilibrium between short lived protein conformations. The Zimm-Bragg theory is an ideal algorithm to handle cooperative processes. Here, we extend the analytical capabilities of the Zimm-Bragg theory in two directions. First, we combine the Zimm-Bragg partition function Z(T) with statistical-mechanical thermodynamics, explaining the thermodynamic system properties enthalpy, entropy and free energy with molecular parameters only. Second, the molecular enthalpy h0 to unfold a single amino acid residue is made temperature-dependent. The addition of a heat capacity term cv allows predicting not only heat denaturation, but also cold denaturation. Moreover, it predicts the heat capacity increase [Formula] in protein unfolding. The theory is successfully applied to differential scanning calorimetry experiments of proteins of different size and structure, that is, gpW62 (62aa), ubiquitin (74aa), lysozyme (129aa), metmyoglobin (153aa) and mAb monoclonal antibody (1290aa). Particular attention was given to the free energy, which can easily be obtained from the heat capacity Cp(T). The DSC experiments reveal a zero free energy for the native protein with an immediate decrease to negative free energies upon cold and heat denaturation. This trapezoidal shape is precisely reproduced by the Zimm-Bragg theory, whereas the so far applied non-cooperative 2-state model predicts a parabolic shape with a positive free energy maximum of the native protein. We demonstrate that the molecular parameters of the Zimm-Bragg theory have a well-defined physical meaning. In addition to predicting protein stability, independent of protein size, they yield estimates of unfolding kinetics and can be connected to molecular dynamics calculations.

biophysics↗

bioRxiv2019

Islet vascularization is regulated by primary endothelial cilia via VEGF-A dependent signaling

RationaleAccumulating evidence point to a role for primary cilia in endothelial cell function. Islet vascularization is an important determinant of islet function and glucose homeostasis. We have previously shown that {beta}-cell cilia directly regulate insulin secretion. However, it is unclear whether primary cilia are also implicated in islet vascularization and thus contribute to glucose homeostasis.\n\nObjectiveTo characterize the role of primary cilia in islet vascularization.\n\nMethods and ResultsAt four weeks, Bbs4-/- islets show markedly lower intra-islet capillary density with enlarged diameters. We transplanted islets into the anterior chamber (ACE) of mouse eyes for longitudinal and non-invasive in vivo monitoring of vascular morphology. Bbs4-/- islets exhibited significantly delayed re-vascularization and enlarged vessels during engraftment. Similar vascular phenotypes were observed in two other ciliopathy models. By shifting the relative contributions of host versus donor endothelial cells in islet revascularization, we found that primary cilia on endothelial cells is essential for this process. Electron microscopy analysis further revealed a lack of fenestration in engrafted Bbs4-/- islets, partially impairing vascular permeability and glucose delivery to {beta}-cells. Finally, we identified that Vascular endothelial cell growth factor A (VEGF-A)/VEGF receptor 2 (VEGFR2) signalling is involved in islet vascularization, islet function and vascular fenestration. In vitro silencing of two different ciliary genes in endothelial cells disrupts VEGF-A/ VEGFR2 internalization and phospho-activation of downstream signalling components. Consequently, key features of angiogenesis including proliferation, migration and tube formation are attenuated in BBS4 silenced endothelial cells.\n\nConclusionsEndothelial cell primary cilia regulate islet vascularization and vascular barrier function via VEGF-A/ VEGFR2 signaling pathway. Islet vascularization is impaired in four weeks old Bbs4-/- mice. Long-time monitoring of re-vascularization of WT and Bbs4-/- islets recapitulates the phenotype and demonstrates a role for cilia in islet vascularization and vascular barrier function. VEGF-A/ VEGFR2-dependent signalling is regulated by endothelial primary cilia.

physiology↗

bioRxiv2018

In vivo phosphoproteomics reveals pathogenic signaling changes in diabetic islets

Progressive decline of pancreatic beta cells function is key to the pathogenesis of type 2 diabetes. Protein phosphorylation is the central mechanism controlling glucose-stimulated insulin secretion in beta cells. However, if and how signaling networks are remodeled in diabetic islets in vivo remain unknowns. Here we applied high-sensitivity mass spectrometry-based proteomics and quantified the levels of about 6,500 proteins and 13,000 phosphopeptides in islets of obese diabetic mice and matched controls. This highlighted drastic remodeling of key kinase hubs and signaling pathways. We integrated our phosphoproteomic dataset with a literature-derived signaling network, which revealed a crucial and conserved role of GSK3 kinase in the control of the beta cells-specific transcription factor PDX1 and insulin secretion, which we functionally verified. Our resource will enable the community to investigate potential mechanisms and drug targets in type 2 diabetes.

systems biology↗