Effect of macromolecular crowding on the kinetics of glycolytic enzymes and the behaviour of glycolysis in yeast

Water is involved in all aspects of biological activity, both as a solvent and as a reactant. It is hypothesized that intracellular water is in a highly structured state due to the high concentrations of macromolecules in the cell and that this may change the activity of intracellular enzymes due to altered binding affinities and allosteric regulations. Here we first investigate the kinetics of two glycolytic enzymes in artificially crowded aqueous solutions and show that crowding does indeed change their kinetics. Based on our kinetic measurements we propose a new model of oscillating glycolysis that instead of Michaelis-Menten or Monod-Wyman-Changeux kinetics uses the Yang-Ling adsorption isotherm introduced by G. Ling in the frame of the Association-Induction (AI) hypothesis. Using this model, we can reproduce previous experimental observations of the coupling of glycolytic oscillations and intracellular water dynamics, e.g., (i) during the metabolic oscillations, the latter variable oscillates in phase with ATP activity, and (ii) the emergence of glycolytic oscillations largely depends on the extent of intracellular water dipolar relaxation in cells in the resting state. Our results support the view that the extent of intracellular water dipolar relaxation is regulated by the ability of cytoplasmic proteins to polarize intracellular water with the assistance of ATP, as suggested in the AI hypothesis. This hypothesis may be relevant to the interpretation of many other biological oscillators, including cell signalling processes.Fil: Thoke, Henrik S.. University Of Southern Denmark; DinamarcaFil: Bagatolli, Luis Alberto. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Córdoba. Instituto de Investigación Médica Mercedes y Martín Ferreyra. Universidad Nacional de Córdoba. Instituto de Investigación Médica Mercedes y Martín Ferreyra; ArgentinaFil: Olsen, Lars F.. University Of Southern Denmark; Dinamarc

Impact of macromolecular crowding on the mesomorphic behavior of lipid self-assemblies

Using LAURDAN fluorescence we observed that water dynamics measured at the interface of DOPC bilayers can be differentially regulated by the presence of crowded suspensions of different proteins (HSA, IgG, Gelatin) and PEG, under conditions where the polymers are not in direct molecular contact with the lipid interface. Specifically, we found that the decrease in water dipolar relaxation at the membrane interface correlates with an increased fraction of randomly oriented (or random coil) configurations in the polymers, as Gelatin > PEG > IgG > HSA. By using the same experimental strategy, we also demonstrated that structural transitions from globular to extended conformations in proteins can induce transitions between lamellar and non-lamellar phases in mixtures of DOPC and monoolein. Independent experiments using Raman spectroscopy showed that aqueous suspensions of polymers exhibiting high proportions of randomly oriented conformations display increased fractions of tetracoordinated water, a configuration that is dominant in ice. This indicates a greater capacity of this type of structure for polarizing water and consequently reducing its chemical activity. This effect is in line with one of the tenets of the Association Induction Hypothesis, which predicts a long-range dynamic structuring of water molecules via their interactions with proteins (or other polymers) showing extended conformations. Overall, our results suggest a crucial role of water in promoting couplings between structural changes in macromolecules and supramolecular arrangements of lipids. This mechanism may be of relevance to cell structure/function when the crowded nature of the intracellular milieu is considered.Fil: Mangiarotti, Agustín. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Córdoba. Instituto de Investigación Médica Mercedes y Martín Ferreyra. Universidad Nacional de Córdoba. Instituto de Investigación Médica Mercedes y Martín Ferreyra; ArgentinaFil: Bagatolli, Luis Alberto. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Córdoba. Instituto de Investigación Médica Mercedes y Martín Ferreyra. Universidad Nacional de Córdoba. Instituto de Investigación Médica Mercedes y Martín Ferreyra; Argentina. Universidad Nacional de Córdoba. Facultad de Ciencias Químicas. Departamento de Química Biológica; Argentin

Lipids, membranes, colloids and cells: A long view

This paper revisits long-standing ideas about biological membranes in the context of an equally long-standing, but hitherto largely unappreciated, perspective of the cell based on concepts derived from the physics and chemistry of colloids. Specifically, we discuss important biophysical aspects of lipid supramolecular structure to understand how the intracellular milieu may constrain lipid self-assembly. To this end we will develop four lines of thought: first, we will look at the historical development of the current view of cellular structure and physiology, considering also the plurality of approaches that influenced its formative period. Second, we will review recent basic research on the structural and dynamical properties of lipid aggregates as well as the role of phase transitions in biophysical chemistry and cell biology. Third, we will present a general overview of contemporary studies into cellular compartmentalization in the context of a very rich and mostly forgotten general theory of cell physiology called the Association-Induction Hypothesis, which was developed around the time that the current view of cells congealed into its present form. Fourth, we will examine some recent developments in cellular studies, mostly from our laboratory, that raise interesting issues about the dynamical aspects of cell structure and compartmentalization. We will conclude by suggesting what we consider are relevant questions about the nature of cellular processes as emergent phenomena.Fil: Bagatolli, Luis Alberto. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Córdoba. Instituto de Investigación Médica Mercedes y Martín Ferreyra. Universidad Nacional de Córdoba. Instituto de Investigación Médica Mercedes y Martín Ferreyra; Argentina. Memphys - International And Interdisciplinary Research Network; Dinamarca. Universidad Nacional de Córdoba. Facultad de Ciencias Exactas, Físicas y Naturales. Departamento de Química. Cátedra de Química Biológica; ArgentinaFil: Stock, Roberto. Memphys - International And Interdisciplinary Research Network; Dinamarc

Coupled response of membrane hydration with oscillating metabolism in live cells: An alternative way to modulate structural aspects of biological membranes?

We propose that active metabolic processes may regulate structural changes in biological membranes via the physical state of cell water. This proposition is based on recent results obtained from our group in yeast cells displaying glycolytic oscillations, where we demonstrated that there is a tight coupling between the oscillatory behavior of glycolytic metabolites (ATP, NADH) and the extent of the dipolar relaxation of intracellular water, which oscillates synchronously. The mechanism we suggest involves the active participation of a polarized intracellular water network whose degree of polarization is dynamically modulated by temporal ATP fluctuations caused by metabolism with intervention of a functional cytoskeleton, as conceived in the long overlooked association-induction hypothesis (AIH) of Gilbert Ling. Our results show that the polarized state of intracellular water can be propagated from the cytosol to regions containing membranes. Since changes in the extent of the polarization of water impinge on its chemical activity, we hypothesize that metabolism dynamically controls the local structure of cellular membranes via lyotropic effects. This hypothesis offers an alternative way to interpret membrane related phenomena (e.g., changes in local curvature pertinent to endo/exocytosis or dynamical changes in membranous organelle structure, among others) by integrating relevant but mostly overlooked physicochemical characteristics of the cellular milieu.Fil: Bagatolli, Luis Alberto. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Córdoba. Instituto de Investigación Médica Mercedes y Martín Ferreyra. Universidad Nacional de Córdoba. Instituto de Investigación Médica Mercedes y Martín Ferreyra; ArgentinaFil: Stock, Roberto P.. International and Interdisciplinary Research Network; DinamarcaFil: Olsen, Lars F.. University Of Southern Denmark; Dinamarc

Análisis filosófico sobre la concepción neurocientífica de los rasgos autistas: aportes desde el normativismo vital de Georges Canguilhem

Taking Georges Canguilhem’s 1943 book The normal and the pathological as a starting point, this article explores the ways in which the neurosciences define, validate, and legitimize the existence of autistic traits as a subclinical expression of autism. The general hypothesis is that different assumptions based on a naturalistic perspective of health and disease have become consolidated in the specialized literature. Such assumptions include that behaviors should be explained strictly in biological terms, that there is an objective and statistical parameter of normality, and that individuals’ behaviors can be analyzed independently of their context. Based on some aspects of Canguilhem’s arguments regarding health, normality, and normativity, we analyze the ways in which the neurosciences assign a quasi-pathological quality to the descriptive notion of autistic traits. It is possible to conclude that this process involves certain assumptions taken in an aprioristic and uncritical manner, which inhibits the discussion of key aspects associated with the nature of autism and that end up pathologizing differences between people.A partir de la obra Lo normal y lo patológico de Georges Canguilhem, publicada en 1943, se explora cómo las neurociencias definen y legitiman, en la actualidad, la existencia de los rasgos autistas como expresión subclínica de autismo. La hipótesis general del presente trabajo es que en la bibliografía especializada de este ámbito parecen consolidarse diferentes supuestos asumidos en el marco de una perspectiva naturalista de la salud. Tales supuestos refieren a que los comportamientos deben ser explicados estrictamente en términos biológicos, que existe un parámetro de normalidad de carácter objetivo y estadístico, y que las conductas de los individuos pueden ser analizadas separadamente del contexto. Retomando algunos aspectos de la propuesta de Canguilhem sobre salud, normalidad y normatividad, se analiza de qué manera las neurociencias otorgan una cualidad cuasi-patológica a una noción descriptiva como la de rasgos autistas. A nuestro entender, este proceso involucra la asunción apriorística y acrítica de supuestos que impiden poner en discusión aspectos elementales asociados a la naturaleza del autismo y que conducen a patologizar la diferencia entre personas

The cell as a gel: materials for a conceptual discussion

Recent results from our laboratory support the view that the intracellular milieu cannot be treated as a homogeneous dilute system and, more importantly, reveal for the first time a dynamical coupling between intracellular water and an active metabolic process involving fluctuations in ATP concentration. These results are difficult to understand in light of the premises that currently underpin the description of the function of cellular systems, e.g. van’t Hoff’s ideal solution theory, diffusion and mass action kinetics. Particularly, they emphasize the need to incorporate features of the cell interior that have been largely overlooked in the dominant model of the cell, such as crowding and limited availability of free water. This article discusses this problem by reconsidering an alternate view, called the association-induction hypothesis, which emphasizes the relevance of emergent properties of the cell cytosol during cellular function. This hypothesis provides a very reasonable theoretical framework to explain recently reported observations about the dynamical coupling of mechanochemical (i.e. viscoelastic) properties of the cell cytoplasm and cellular chemical transformations (metabolism).Los resultados experimentales obtenidos recientemente en nuestro laboratorio apoyan la idea que el medio intracelular no puede ser tratado como un sistema homogéneo (o solución diluida), revelando además por primera vez un acoplamiento dinámico entre el comportamiento colectivo del agua intracelular y un proceso metabólico activo que muestra fluctuaciones en la concentración de ATP. Estos nuevos resultados -que son difíciles de interpretar en base a los supuestos más generalmente utilizados para interpretar las bases fisicoquímicas de la fisiología de los sistemas celulares (p.ej. teoría de las soluciones ideales de van't Hoff, difusión, y cinética de acción de masas)- subrayan la necesidad urgente de incorporar características importantes del interior celular, tales como el hacinamiento molecular y la escasa disponibilidad de agua libre. Este artículo analiza críticamente este problema considerando una hipótesis alternativa, llamada hipótesis de asociación-inducción, la cual hace hincapié en la importancia de las propiedades emergentes del citosol durante la función celular. Esta hipótesis proporciona un marco teórico razonable para explicar nuestras observaciones, particularmente el acoplamiento dinámico entre las propiedades mecanoquímicas (o viscoelásticas) del citoplasma celular y las transformaciones químicas (metabolismo) en el interior celular.Sociedad Argentina de Fisiologí

Dengue and Zika virus capsid proteins bind to membranes and self-assemble into liquid droplets with nucleic acids

Dengue virus (DENV) and Zika virus (ZIKV) capsid proteins efficiently recruit and surround the viral RNA at the endoplasmic reticulum (ER) membrane to yield nascent viral particles. However, little is known either about the molecular mechanisms by which multiple copies of capsid proteins assemble into nucleocapsids (NCs) or how the NC is recruited and wrapped by the ER membrane during particle morphogenesis. Here, we measured relevant interactions concerning this viral process using purified DENV and ZIKV capsid proteins, membranes mimicking the ER lipid composition, and nucleic acids in in vitro conditions to understand the biophysical properties of the RNA genome encapsidation process. We found that both ZIKV and DENV capsid proteins bound to liposomes at liquid-disordered phase regions, docked exogenous membranes, and RNA molecules. Liquid–liquid phase separation is prone to occur when positively charged proteins interact with nucleic acids, which is indeed the case for the studied capsids. We characterized these liquid condensates by measuring nucleic acid partition constants and the extent of water dipolar relaxation, observing a cooperative process for the formation of the new phase that involves a distinct water organization. Our data support a new model in which capsid–RNA complexes directly bind the ER membrane, seeding the process of RNA recruitment for viral particle assembly. These results contribute to our understanding of the viral NC formation as a stable liquid–liquid phase transition, which could be relevant for dengue and Zika gemmation, opening new avenues for antiviral intervention.Fil: Ambroggio, Ernesto Esteban. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Córdoba. Centro de Investigaciones en Química Biológica de Córdoba. Universidad Nacional de Córdoba. Facultad de Ciencias Químicas. Centro de Investigaciones en Química Biológica de Córdoba; ArgentinaFil: Costa Navarro, Guadalupe Soledad. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario. Instituto de Investigaciones Bioquímicas de Buenos Aires. Fundación Instituto Leloir. Instituto de Investigaciones Bioquímicas de Buenos Aires; ArgentinaFil: Pérez Socas, Luis Benito. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Córdoba. Centro de Investigaciones en Química Biológica de Córdoba. Universidad Nacional de Córdoba. Facultad de Ciencias Químicas. Centro de Investigaciones en Química Biológica de Córdoba; ArgentinaFil: Bagatolli, Luis Alberto. Universidad Nacional de Córdoba. Facultad de Ciencias Químicas. Departamento de Química Biológica; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Córdoba. Instituto de Investigación Médica Mercedes y Martín Ferreyra. Universidad Nacional de Córdoba. Instituto de Investigación Médica Mercedes y Martín Ferreyra; ArgentinaFil: Gamarnik, Andrea Vanesa. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario. Instituto de Investigaciones Bioquímicas de Buenos Aires. Fundación Instituto Leloir. Instituto de Investigaciones Bioquímicas de Buenos Aires; Argentin

Inductive effects in amino acids and peptides: Ionization constants and tryptophan fluorescence

Although inductive effects in organic compounds are known to influence chemical properties such as ionization constants, their specific contribution to the properties/behavior of amino acids and functional groups in peptides remains largely unexplored. In this study we developed a computationally economical algorithm for ab initio calculation of the magnitude of inductive effects for non-aromatic molecules. The value obtained by the algorithm is called the Inductive Index and we observed a high correlation (R2 = 0.9427) between our calculations and the pKa values of the alpha-amino groups of amino acids with non-aromatic side-chains. Using a series of modified amino acids, we also found similarly high correlations (R2 > 0.9600) between Inductive Indexes and two wholly independent chemical properties: i) the pKa values of ionizable side-chains and, ii) the fluorescence response of the indole group of tryptophan. After assessing the applicability of the method of calculation at the amino acid level, we extended our study to tryptophan-containing peptides and established that inductive contributions of neighboring side-chains are transmitted through peptide bonds. We discuss possible contributions to the study of proteins.Fil: Lara Popoca, Jesús. Universidad Nacional Autónoma de México; MéxicoFil: Thoke, Henrik S.. International and Interdisciplinary Research Network; DinamarcaFil: Stock, Roberto P.. Universidad Nacional Autónoma de México; MéxicoFil: Rudino Pinera, Enrique. Universidad Nacional Autónoma de México; MéxicoFil: Bagatolli, Luis Alberto. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Córdoba. Instituto de Investigación Médica Mercedes y Martín Ferreyra. Universidad Nacional de Córdoba. Instituto de Investigación Médica Mercedes y Martín Ferreyra; Argentin

Tight Coupling of Metabolic Oscillations and Intracellular Water Dynamics in <i>Saccharomyces cerevisiae</i>

We detected very strong coupling between the oscillating concentration of ATP and the dynamics of intracellular water during glycolysis in Saccharomyces cerevisiae. Our results indicate that: i) dipolar relaxation of intracellular water is heterogeneous within the cell and different from dilute conditions, ii) water dipolar relaxation oscillates with glycolysis and in phase with ATP concentration, iii) this phenomenon is scale-invariant from the subcellular to the ensemble of synchronized cells and, iv) the periodicity of both glycolytic oscillations and dipolar relaxation are equally affected by D2O in a dose-dependent manner. These results offer a new insight into the coupling of an emergent intensive physicochemical property of the cell, i.e. cell-wide water dipolar relaxation, and a central metabolite (ATP) produced by a robustly oscillating metabolic process