On the rational design of cooperative receptors

Cooperativity (homotropic allostery) is the primary mechanism by which evolution steepens the binding curves of biomolecular receptors to produce more responsive input-output behavior in biomolecular systems. Motivated by the ubiquity with which nature employs this effect, over the past 15 years we, together with other groups, have engineered this mechanism into several otherwise noncooperative receptors. These efforts largely aimed to improve the utility of such receptors in artificial biotechnologies, such as synthetic biology and biosensors, but they have also provided the first quantitative, experimental tests of longstanding ideas about the mechanisms underlying cooperativity. In this article, we review the literature on the design of this effect, paying particular attention to the design strategies involved, the extent to which each can be rationally applied to (and optimized for) new receptors, and what each teaches us about the origins and optimization of this important phenomenon

Beyond the two-state model of switching in biology and computation

The thesis presents various perspectives on physical and biological computation. Our fundamental object of study in both these contexts is the notion of switching/erasing a bit. In a physical context, a bit is represented by a particle in a double well, whose dynamics is governed by the Langevin equation. We define the notions of reliability and erasing time-scales in addition to the work required to erase a bit for a given family of control protocols. We call bits “optimal” if they meet the required reliability and erasing time requirements with minimal work cost. We find that optimal bits always saturate the erasing time requirement, but may not saturate the reliability time requirement. This allows us to eliminate several regions of parameter space as sub-optimal. In a biological context, our bits are represented by substrates that are acted upon by catalytic enzymes. We define retroactivity as the back-signal propagated by the downstream system when connected to the upstream system. We analyse certain upstream systems that can help mitigate retroactivity. However, these systems require a substantial pool of resources and are therefore not optimal. As a consequence, we turn our attention to insulating networks called push-pull motifs. We find that high rates of energy consumption are not essential to alleviate retroactivity in push-pull motifs; all we need is to couple weakly to the upstream system. However, this approach is not resilient to cross-talk caused by leak reactions in the circuit. Next, we consider a single enzyme-substrate reaction and analyse its mechanism. Our system has two intermediate states (enzyme-substrate complexes). Our main question is “How should we choose binding energies of the intermediates to minimize sequestra- tion of substrates (retroactivity), whilst maintaining a minimum flux at steady-state?”. Choosing very low binding energies increases retroactivity since the system spends a considerable proportion of time in the intermediate states. Choosing binding energies that are very high reduces retroactivity, but hinders the progress of the reaction. As a result, we find that the the optimal binding energies are both moderate, and indeed tuned with each other. In particular, their difference is related to the free energy difference between the products and reactants.Open Acces

Divergence, Recombination and Retention of Functionality During Protein Evolution

We have only a vague idea of precisely how protein sequences evolve in the context of protein structure and function. This is primarily because structural and functional contexts are not easily predictable from the primary sequence, and evaluating patterns of evolution at individual residue positions is also difficult. As a result of increasing biodiversity in genomics studies, progress is being made in detecting context-dependent variation in substitution processes, but it remains unclear exactly what context-dependent patterns we should be looking for. To address this, we have been simulating protein evolution in the context of structure and function using lattice models of proteins and ligands (or substrates). These simulations include thermodynamic features of protein stability and population dynamics. We refer to this approach as \u27ab initio evolution\u27 to emphasise the fact that the equilibrium details of fitness distributions arise from the physical principles of the system and not from any preconceived notions or arbitrary mathematical distributions. Here, we present results on the retention of functionality in homologous recombinants following population divergence. A central result is that protein structure characteristics can strongly influence recombinant functionality. Exceptional structures with many sequence options evolve quickly and tend to retain functionality--even in highly diverged recombinants. By contrast, the more common structures with fewer sequence options evolve more slowly, but the fitness of recombinants drops off rapidly as homologous proteins diverge. These results have implications for understanding viral evolution, speciation and directed evolutionary experiments. Our analysis of the divergence process can also guide improved methods for accurately approximating folding probabilities in more complex but realistic systems

Conformational Flexibility Drives Cold Adaptation in Pseudoalteromonas haloplanktis TAC125 Globins

Significance: Temperature is one of the most important drivers in shaping protein adaptations. Many biochemical and physiological processes are influenced by temperature. Proteins and enzymes from organisms living at low temperature are less stable in comparison to high-temperature adapted proteins. The lower stability is generally due to greater conformational flexibility. Recent Advances: Adaptive changes in the structure of cold-adapted proteins may occur at subunit interfaces, distant from the active site, thus producing energy changes associated with conformational transitions transmitted to the active site by allosteric modulation, valid also for monomeric proteins in which tertiary structural changes may play an essential role. Critical Issues: Despite efforts, the current experimental and computational methods still fail to produce general principles on protein evolution, since many changes are protein and species dependent. Environmental constraints or other biological cellular signals may override the ancestral information included in the structure of the protein, thus introducing inaccuracy in estimates and predictions on the evolutionary adaptations of proteins in response to cold adaptation. Future Directions: In this review, we describe the studies and approaches used to investigate stability and flexibility in the cold-adapted globins of the Antarctic marine bacterium Pseudoalteromonas haloplanktis TAC125. In fact, future research directions will be prescient on more detailed investigation of cold-adapted proteins and the role of fluctuations between different conformational states.Fil: Giordano, Daniela. Institute Of Biosciences And Bioresources; ItaliaFil: Boubeta, Fernando Martín. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Ciudad Universitaria. Instituto de Química, Física de los Materiales, Medioambiente y Energía. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Instituto de Química, Física de los Materiales, Medioambiente y Energía; ArgentinaFil: di Prisco, Guido. Institute Of Biosciences And Bioresources; ItaliaFil: Estrin, Dario Ariel. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Ciudad Universitaria. Instituto de Química, Física de los Materiales, Medioambiente y Energía. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Instituto de Química, Física de los Materiales, Medioambiente y Energía; ArgentinaFil: Smulevich, Giulietta. Firenze University; ItaliaFil: Viappiani, Christiano. Università di Parma; ItaliaFil: Verde, Cinzia. Institute Of Biosciences And Bioresources; Itali

Colloid osmotic parameterization and measurement of subcellular crowding

© The Author(s), 2019. This article is distributed under the terms of the Creative Commons Attribution License. The definitive version was published in Mitchison, T. J. (2019). Colloid osmotic parameterization and measurement of subcellular crowding. Molecular Biology of the Cell, 30(2), (2019): 173-180, doi:10.1091/mbc.E18-09-0549.Crowding of the subcellular environment by macromolecules is thought to promote protein aggregation and phase separation. A challenge is how to parameterize the degree of crowding of the cell interior or artificial solutions that is relevant to these reactions. Here I review colloid osmotic pressure as a crowding metric. This pressure is generated by solutions of macromolecules in contact with pores that are permeable to water and ions but not macromolecules. It generates depletion forces that push macromolecules together in crowded solutions and thus promotes aggregation and phase separation. I discuss measurements of colloid osmotic pressure inside cells using the nucleus, the cytoplasmic gel, and fluorescence resonant energy transfer (FRET) biosensors as osmometers, which return a range of values from 1 to 20 kPa. I argue for a low value, 1–2 kPa, in frog eggs and perhaps more generally. This value is close to the linear range on concentration–pressure curves and is thus not crowded from an osmotic perspective. I discuss the implications of a low crowding pressure inside cells for phase separation biology, buffer design, and proteome evolution. I also discuss a pressure–tension model for nuclear shape, where colloid osmotic pressure generated by nuclear protein import inflates the nucleus.This article was prompted by lively discussions at the Marine Biological Laboratory (MBL) Physiology Course, Woods Hole, MA. I particularly thank Annie Pipathsouk (University of California, San Franscico) and Charlotte Strandkvist (Harvard Medical School) for experimental work in frog egg extract; James Pelletier (MIT), Tony Hyman (MPI Dresden), and Rob Phillips (Cal. Tech.) for discussions; and Nikon for microscopy support at MBL. T.J.M. is supported by National Institute of General Medical Sciences 39565

Molecular Modeling in Enzyme Design, Toward In Silico Guided Directed Evolution

Directed evolution (DE) creates diversity in subsequent rounds of mutagenesis in the quest of increased protein stability, substrate binding, and catalysis. Although this technique does not require any structural/mechanistic knowledge of the system, the frequency of improved mutations is usually low. For this reason, computational tools are increasingly used to focus the search in sequence space, enhancing the efficiency of laboratory evolution. In particular, molecular modeling methods provide a unique tool to grasp the sequence/structure/function relationship of the protein to evolve, with the only condition that a structural model is provided. With this book chapter, we tried to guide the reader through the state of the art of molecular modeling, discussing their strengths, limitations, and directions. In addition, we suggest a possible future template for in silico directed evolution where we underline two main points: a hierarchical computational protocol combining several different techniques and a synergic effort between simulations and experimental validation.Peer ReviewedPostprint (author's final draft

Física de procesos celulares : el papel de las escalas espaciales características de la membrana celula

Tesis de la Universidad Complutense de Madrid, Facultad de Ciencias Físicas, Departamento de Estructura de la Materia, Física Térmica y Electrónica, leída el 30-11-2018The study of cells helps us understand how organisms function. After all, our bodies are made up of trillions of cells. There are many processes that cells must complete to carry out their life functions and to meet their basic needs. In the present thesis, we will study the physics of some cellular processes in which the effect of cell size and membrane curvature plays a major role. First, we will investigate the mechanics of constriction during cell division. Cell division is the process of formation of new daughter cells from the pre‐existing mother cell. An important phase preceding division is cell constriction, a non‐spontaneous process which involves large membrane deformations at the site of fission. In this thesis, we will investigate the mechanical route for symmetric constriction using a model cell composed by a flexible membrane that encloses the cytoplasm...El estudio de las células nos ayuda a comprender cómo funcionan los organismos. Al fin y al cabo, nuestros cuerpos están formados por billones de células. Hay muchos procesos que las células deben completar para llevar a cabo sus funciones vitales y satisfacer sus necesidades. En la presente tesis, estudiaremos la física de algunos procesos en los que el efecto del tamaño celular y la curvatura de la membrana es crucial.Primero, investigaremos la mecánica de la constricción durante la división celular. La división celular es el proceso de formación de células hijas a partir de la célula madre. Una fase importante que precede a la división es la constricción celular, un proceso no espontáneo que involucra grandes deformaciones de membrana en la zona de fisión. En esta tesis estudiaremos el mecanismo de la constricción simétrica usando como célula modelo una membrana flexible que encierra el citoplasma..Depto. de Estructura de la Materia, Física Térmica y ElectrónicaFac. de Ciencias FísicasTRUEunpu

Coarse-Grained Mean-Field Simulations of Surfactant Micelles: Static and Dynamic Equilibrium Properties

Les molècules de surfactants estan compostes per seccions hidrofíliques i hidrofòbiques del qual les interaccions oposades amb el medi solvent deriven en un comportament particular on, a una concentració de surfactant coneguda com la concentració crítica micel·lar (CMC), els surfactants s'associen en micel·les. No obstant això, malgrat el gran nombre de treballs de simulació, experimentals i teòrics, sembla estar encara incomplet l'enteniment a nivell microscòpic de l'acte assemblage de surfactants en agregats micelars. A la recerca d'un millor enteniment del procés de micelización i el seu impacte en propietats microscòpiques i macroscòpiques, aquest treball fa servir el mètode single-chain mean-field (SCMF) per a un grup de sistemes de surfactants representats per una sèrie de models de grans. En particular, el treball aborda: (i) l'estudi de propietats estàtiques d'equilibri per a diferents sistemes de surfactants i l'anàlisi de desviacions experimentals de propietats d'equilibri per surfactants gemini del qual les escales de longitud disminueixen la possibilitat de ser estudiats pels mètodes de simulació usuals, i, (ii) el desenvolupament d'una versió dinàmica del SCMF per obtenir propietats dinàmiques en equilibri que ens permetin estudiar el fenomen d'intercanvi cinètic en sistemes micelars.Las moléculas de surfactantes están compuestas por secciones hidrofílicas e hidrofóbicas cuyas interacciones opuestas con el medio solvente derivan en un comportamiento particular donde, a una concentración de surfactante conocida como la concentración crítica micelar (CMC), los surfactants se asocian en micelas. No obstante, a pesar del gran número de trabajos de simulación, experimentales y teóricos, parece estar aún incompleto el entendimiento a nivel microscópico del auto ensamblaje de surfactantes en agregados micelares. En busca de un mejor entendimiento del proceso de micelización y su impacto en propiedades microscópicas y macroscópicas, este trabajo usa el método single-chain mean-field (SCMF) para un grupo de sistemas de surfactantes representados por una serie de modelos de granos. En particular, el trabajo aborda: (i) el estudio de propiedades estáticas de equilibrio para diferentes sistemas de surfactantes y el análisis de desviaciones experimentales de propiedades de equilibrio para surfactantes gemini cuyas escalas de longitud disminuyen la posibilidad de ser estudiados por los métodos de simulación usuales, y, (ii) el desarrollo de una versión dinámica del SCMF para obtener propiedades dinámicas en equilibrio que nos permitan estudiar el fenómeno de intercambio cinético en sistemas micelares.Surfactants molecules are composed by hydrophilic and hydrophobic moieties whose opposite interactions with the solvent medium result in a particular behavior where, at a surfactant concentration known as the critical micelle concentration (CMC), the surfactants self-associate into micelles. However, despite the wide number of simulation, experimental and theoretical works, microscopic understanding of the self-assembly of surfactants into micellar aggregates seems to be still incomplete. Looking for a deep knowledge of the micellization process and its impact on microscopic and macroscopic properties, this work is aimed to use the single-chain mean-field (SCMF) theory for a diverse set of surfactant systems represented by a series of coarse-grained models. In particular, the work coverages the following: (i) the study of static equilibrium properties of different surfactant systems and the analysis of experimental deviations of equilibrium properties for gemini surfactant systems whose length scales decrease the possibility of being studied by regular simulation methods, and, (ii) development of a dynamic version of the SCMF scheme, to obtain dynamic equilibrium properties that enable us to study kinetics exchange phenomena in micellar systems