How Water's Properties Are Encoded in Its Molecular Structure and Energies.

How are water's material properties encoded within the structure of the water molecule? This is pertinent to understanding Earth's living systems, its materials, its geochemistry and geophysics, and a broad spectrum of its industrial chemistry. Water has distinctive liquid and solid properties: It is highly cohesive. It has volumetric anomalies-water's solid (ice) floats on its liquid; pressure can melt the solid rather than freezing the liquid; heating can shrink the liquid. It has more solid phases than other materials. Its supercooled liquid has divergent thermodynamic response functions. Its glassy state is neither fragile nor strong. Its component ions-hydroxide and protons-diffuse much faster than other ions. Aqueous solvation of ions or oils entails large entropies and heat capacities. We review how these properties are encoded within water's molecular structure and energies, as understood from theories, simulations, and experiments. Like simpler liquids, water molecules are nearly spherical and interact with each other through van der Waals forces. Unlike simpler liquids, water's orientation-dependent hydrogen bonding leads to open tetrahedral cage-like structuring that contributes to its remarkable volumetric and thermal properties

Molecular Dynamics Simulations using Advanced Sampling and Polarizable Force Fields

Molecular dynamics (MD) simulations were carried out for aqueous dipeptides, water over self-assembled monolayer (SAM) surfaces, and the nicotinic acetylcholine receptor (nAChR) ion channel. The main goal is to use advanced methods to increase the accuracy of molecular dynamics simulations while seeking solutions to problems relevant to chemistry, biophysics and materials science. In addition, activation energies of several cyclodimerization reactions were studied quantum mechanically. The simulations of the aqueous dipeptides and SAM surfaces involve modeling and detailed analysis of interfacial water, which is of interest to a range of fields from biology to materials science. For example, water has a central role in biology and medicine since biomolecules cannot function without water. Both sets of simulations were performed using both polarizable and nonpolarizable force fields. These systems were used as a test ground to assess the effects of explicit incorporation of polarizability and also to determine whether the models can adequately reproduce the experimental data, in particular, the aggregation data of aqueous dipeptides and contact angles of water over SAMs of different chemical character. Since the systems are well-characterized and relatively simple, they provide excellent models to test polarizable force fields to increase the accuracy of molecular dynamics simulations. Polarizable water was depolarized around dipeptide solutes and also at the interface with different SAM surfaces, reflecting its ability to adapt to heterogeneous electrostatic environments. Although the water shows more realistic structure and dynamics in the polarizable simulations, the peptide aggregation behavior agrees less well with the experiment. In this case, neither model successfully reproduces the experimental degree of aggregation. In the case of SAM surfaces, both sets of simulations produce fairly similar results. More studies are suggested to further test and improve the polarizable force fields. The third system studied is the modeling of wild-type and mutant nAChR ion channel proteins. Adaptive biasing force method was used to achieve improved sampling, and subsequently increase the efficiency and accuracy of MD simulations. The nAChR channels are involved in a number of cognitive and brain functions including learning and memory. Dysfunction in these receptors are associated in a variety of neuronal diseases including epilepsy, schizophrenia and Alzheimer\u27s Disease. The present study models the wild-type and two physiologically-relevant mutant structures to assess the effects of mutations on ion translocation energetics and the geometry of the channel. Open channel (conducting, active) structures were obtained from the available closed channel structure. One of the mutants was found to increase the energetic barrier for ion translocation, while the other one decreased the barrier. The ion channel structures were analyzed in detail to understand the structural changes that took place during the channel opening. The channel opening was found to be mediated by large-scale helix motions rather than small-scale side chain motions. Aside from the MD simulations, the final project involves quantum mechanical simulations, which are often needed in parametrization of molecular dynamics force fields. Density functional theory (DFT) calculations were employed to calculate the activation energies of three cyclodimerization reactions of trifluorovinyl ether monomers. The results agree with and further explain the experimentally observed reactivity in these types of reactions

Probes of tocopherol biochemistry: fluorophores, imaging agents, and fake antioxidants

The body has many defence systems against reactive radical species, but none are as crucial in the protection of lipid membranes as vitamin E. As a result of a selection process mediated by the α-tocopherol transfer protein (α-TTP), α-tocopherol is the only form of vitamin E retained in the body. This chaperon protein has been well studied because of its role in vitamin E transport. Furthermore, malfunctions of α-TTP cause vitamin E deficiency leading to ataxia and other neurodegenerative disease. Protection of neuronal tissue is critical and is reflected in the high retention of α-tocopherol in the central nervous system. Neuronal tissues receive α tocopherol from astrocytes, cells that are linked to hepatic tissue and able to express α-TTP, however the exact path of delivery between these cells is still unclear. A technique called fluorescent microscopy allows the tracking of fluorescent molecules in cells to find their location and interactions with other parts of the cell. The focus of this study is the synthesis of a fluorescent tocopherol analogue with a long absorption wavelength, high photostability, and that binds selectively to  α-TTP with high affinity. Most health benefits associated with vitamin E consumption are based on its capability to inhibit lipid peroxidation in cell membranes by scavenging reactive oxygen species (ROS). Oxidative damage in membranes puts cells in a “stressful” state, activating signalling events that trigger apoptosis. Vitamin E down-regulates apoptotic functions like inflammation, macrophage activation and cell arrest in a stressed state, returning the cell back to normal functioning. At the same time, vitamin E has a preventive effect for atherosclerosis, Alzheimer’s and cancer. With the deeper understanding of cell signalling processes associated with vitamin E the question arose whether protein interactions or the ROS scavenging is responsible for cell survival. To test this hypothesis, a non-antioxidant but α-TTP binding tocopherol analogue was synthesized and administered into oxidatively stressed, α-TTP deficient cells. If the cells were unable to restore homeostasis and stop apoptosis with the new molecule, this would suggest that the antioxidant function of α-tocopherol is the reason for survival. Cancer is regarded as one of the most detrimental diseases with a high mortality rate. One key aspect in medical research is the increased drug specificity towards targeting cancer. Chemotherapy applies cytotoxic compounds, which weaken the immune system because both malignant and healthy cells are destroyed. The specificity of the anti-cancer drugs are enhanced when encapsulated into liposomes that bear target-directing molecules such as antibodies which recognize cancer cell specific antigens on the cell membrane. The question remains if the encapsulated drug reaches the cancer or not. Magnetic resonance imaging (MRI) and computed tomography (CT) are used to find malignant tissue in the body. CT imaging uses highly charged X-ray particles to scan the patient, possibly having damaging cytotoxic effects. Obtaining MRI results require the use of contrast agents to enhance the quality of images. These agents are based on transition metals, which potentially have chronic toxicity when retained in the body. Alternatively short-lived radiotracers that emit a γ-photon upon positron decay are used through a process called positron emission tomography (PET). Rapid decay times make the use of PET a less toxic alternative, however the decay products might be toxic to the cell. For this reason a vitamin E based PET agent was created, which produces naturally safe decay products based on known metabolites of vitamin E, useful to track liposomal delivery of chemotherapeutic agents. This work describes the non-radioactive synthetic procedures towards a variety of vitamin E PET analogues. The cytotoxicity of the most promising vitamin E PET tracer was evaluated along with its synthetic byproducts

Cell Membrane Penetration without Pore Formation : Chameleonic Properties of Dendrimers in Response to Hydrophobic and Hydrophilic Environments

The mechanism by which cell-penetrating peptides and antimicrobial peptides cross plasma membranes is unknown, as is how cell-penetrating peptides facilitate drug delivery, mediating the transport of small molecules. Once nondisruptive and nonendocytotic pathways are excluded, pore formation is one of the proposed mechanisms, including toroidal, barrel-stave, or carpet models. Spontaneous pores are observed in coarse-grained simulations and less often in molecular dynamics simulations. While pores are widely assumed and inferred, there is no unambiguous experimental evidence of the existence of pores. Some recent experimental studies contradict the mechanistic picture of pore formation, however, highlighting the possibility of a direct translocation pathway that is both nondisruptive and nonendocytotic. In this work, a model is proposed a model for peptide (linear and dendritic) translocation which does not require the presence of pores and which potentially accords with such experiments. It is suggested that a charged peptide, as it experiences an increasingly hydrophobic environment within the membrane surface, can utilize a proton chain transfer mechanism to shed its protons to counter ions or potentially phospholipid head groups in the membrane skin region, thereby becoming compatible with the hydrophobic interior of the membrane. This increases the likelihood to move into the highly hydrophobic core of the membrane and ultimately reach the opposite leaflet to re-acquire protons again, suggesting a potential "chameleon" mechanism for non-disruptive and non-endocytotic membrane translocation. The molecular dynamics simulations reveal stability of peptide bridges joining two membrane leaflets and demonstrate that this can facilitate cross-membrane transport of small drug molecules.Peer reviewe

Cholinium amino acid-based ionic liquids

Boosted by the simplicity of their synthesis and low toxicity, cholinium and amino acid-based ionic liquids have attracted the attention of researchers in many different fields ranging from computational chemistry to electrochemistry and medicine. Among the uncountable IL variations, these substances occupy a space on their own due to their exceptional biocompatibility that stems from being entirely made by metabolic molecular components. These substances have undergone a rather intensive research activity because of the possibility of using them as greener replacements for traditional ionic liquids. We present here a short review in the attempt to provide a compendium of the state-of-the-art scientific research about this special class of ionic liquids based on the combination of amino acid anions and cholinium cations

Protein-protein interactions: impact of solvent and effects of fluorination

Proteins have an indispensable role in the cell. They carry out a wide variety of structural, catalytic and signaling functions in all known biological systems. To perform their biological functions, proteins establish interactions with other bioorganic molecules including other proteins. Therefore, protein-protein interactions is one of the central topics in molecular biology. My thesis is devoted to three different topics in the field of protein-protein interactions. The first one focuses on solvent contribution to protein interfaces as it is an important component of protein complexes. The second topic discloses the structural and functional potential of fluorine's unique properties, which are attractive for protein design and engineering not feasible within the scope of canonical amino acids. The last part of this thesis is a study of the impact of charged amino acid residues within the hydrophobic interface of a coiled-coil system, which is one of the well-established model systems for protein-protein interactions studies. I. The majority of proteins interact in vivo in solution, thus studies of solvent impact on protein-protein interactions could be crucial for understanding many processes in the cell. However, though solvent is known to be very important for protein-protein interactions in terms of structure, dynamics and energetics, its effects are often disregarded in computational studies because a detailed solvent description requires complex and computationally demanding approaches. As a consequence, many protein residues, which establish water-mediated interactions, are neither considered in an interface definition. In the previous work carried out in our group the protein interfaces database (SCOWLP) has been developed. This database takes into account interfacial solvent and based on this classifies all interfacial protein residues of the PDB into three classes based on their interacting properties: dry (direct interaction), dual (direct and water-mediated interactions), and wet spots (residues interacting only through one water molecule). To define an interaction SCOWLP considers a donor–acceptor distance for hydrogen bonds of 3.2 Å, for salt bridges of 4 Å, and for van der Waals contacts the sum of the van der Waals radii of the interacting atoms. In previous studies of the group, statistical analysis of a non-redundant protein structure dataset showed that 40.1% of the interfacial residues participate in water-mediated interactions, and that 14.5% of the total residues in interfaces are wet spots. Moreover, wet spots have been shown to display similar characteristics to residues contacting water molecules in cores or cavities of proteins. The goals of this part of the thesis were: 1. to characterize the impact of solvent in protein-protein interactions 2. to elucidate possible effects of solvent inclusion into the correlated mutations approach for protein contacts prediction To study solvent impact on protein interfaces a molecular dynamics (MD) approach has been used. This part of the work is elaborated in section 2.1 of this thesis. We have characterized properties of water-mediated protein interactions at residue and solvent level. For this purpose, an MD analysis of 17 representative complexes from SH3 and immunoglobulin protein families has been performed. We have shown that the interfacial residues interacting through a single water molecule (wet spots) are energetically and dynamically very similar to other interfacial residues. At the same time, water molecules mediating protein interactions have been found to be significantly less mobile than surface solvent in terms of residence time. Calculated free energies indicate that these water molecules should significantly affect formation and stability of a protein-protein complex. The results obtained in this part of the work also suggest that water molecules in protein interfaces contribute to the conservation of protein interactions by allowing more sequence variability in the interacting partners, which has important implications for the use of the correlated mutations concept in protein interactions studies. This concept is based on the assumption that interacting protein residues co-evolve, so that a mutation in one of the interacting counterparts is compensated by a mutation in the other. The study presented in section 2.2 has been carried out to prove that an explicit introduction of solvent into the correlated mutations concept indeed yields qualitative improvement of existing approaches. For this, we have used the data on interfacial solvent obtained from the SCOWLP database (the whole PDB) to construct a “wet” similarity matrix. This matrix has been used for prediction of protein contacts together with a well-established “dry” matrix. We have analyzed two datasets containing 50 domains and 10 domain pairs, and have compared the results obtained by using several combinations of both “dry” and “wet” matrices. We have found that for predictions for both intra- and interdomain contacts the introduction of a combination of a “dry” and a “wet” similarity matrix improves the predictions in comparison to the “dry” one alone. Our analysis opens up the idea that the consideration of water may have an impact on the improvement of the contact predictions obtained by correlated mutations approaches. There are two principally novel aspects in this study in the context of the used correlated mutations methodology : i) the first introduction of solvent explicitly into the correlated mutations approach; ii) the use of the definition of protein-protein interfaces, which is essentially different from many other works in the field because of taking into account physico-chemical properties of amino acids and not being exclusively based on distance cut-offs. II. The second part of the thesis is focused on properties of fluorinated amino acids in protein environments. In general, non-canonical amino acids with newly designed side-chain functionalities are powerful tools that can be used to improve structural, catalytic, kinetic and thermodynamic properties of peptides and proteins, which otherwise are not feasible within the use of canonical amino acids. In this context fluorinated amino acids have increasingly gained in importance in protein chemistry because of fluorine's unique properties: high electronegativity and a small atomic size. Despite the wide use of fluorine in drug design, properties of fluorine in protein environments have not been yet extensively studied. The aims of this part of the dissertation were: 1. to analyze the basic properties of fluorinated amino acids such as electrostatic and geometric characteristics, hydrogen bonding abilities, hydration properties and conformational preferences (section 3.1) 2. to describe the behavior of fluorinated amino acids in systems emulating protein environments (section 3.2, section 3.3) First, to characterize fluorinated amino acids side chains we have used fluorinated ethane derivatives as their simplified models and applied a quantum mechanics approach. Properties such as charge distribution, dipole moments, volumes and size of the fluoromethylated groups within the model have been characterized. Hydrogen bonding properties of these groups have been compared with the groups typically presented in natural protein environments. We have shown that hydrogen and fluorine atoms within these fluoromethylated groups are weak hydrogen bond donors and acceptors. Nevertheless they should not be disregarded for applications in protein engineering. Then, we have implemented four fluorinated L-amino acids for the AMBER force field and characterized their conformational and hydration properties at the MD level. We have found that hydrophobicity of fluorinated side chains grows with the number of fluorine atoms and could be explained in terms of high electronegativity of fluorine atoms and spacial demand of fluorinated side-chains. These data on hydration agrees with the results obtained in the experimental work performed by our collaborators. We have rationally engineered systems that allow us to study fluorine properties and extract results that could be extrapolated to proteins. For this, we have emulated protein environments by introducing fluorinated amino acids into a parallel coiled-coil and enzyme-ligand chymotrypsin systems. The results on fluorination effect on coiled-coil dimerization and substrate affinities in the chymotrypsin active site obtained by MD, molecular docking and free energy calculations are in strong agreement with experimental data obtained by our collaborators. In particular, we have shown that fluorine content and position of fluorination can considerably change the polarity and steric properties of an amino acid side chain and, thus, can influence the properties that a fluorinated amino acid reveals within a native protein environment. III. Coiled-coils typically consist of two to five right-handed α-helices that wrap around each other to form a left-handed superhelix. The interface of two α-helices is usually represented by hydrophobic residues. However, the analysis of protein databases revealed that in natural occurring proteins up to 20% of these positions are populated by polar and charged residues. The impact of these residues on stability of coiled-coil system is not clear. MD simulations together with free energy calculations have been utilized to estimate favourable interaction partners for uncommon amino acids within the hydrophobic core of coiled-coils (Chapter 4). Based on these data, the best hits among binding partners for one strand of a coiled-coil bearing a charged amino acid in a central hydrophobic core position have been selected. Computational data have been in agreement with the results obtained by our collaborators, who applied phage display technology and CD spectroscopy. This combination of theoretical and experimental approaches allowed to get a deeper insight into the stability of the coiled-coil system. To conclude, this thesis widens existing concepts of protein structural biology in three areas of its current importance. We expand on the role of solvent in protein interfaces, which contributes to the knowledge of physico-chemical properties underlying protein-protein interactions. We develop a deeper insight into the understanding of the fluorine's impact upon its introduction into protein environments, which may assist in exploiting the full potential of fluorine's unique properties for applications in the field of protein engineering and drug design. Finally we investigate the mechanisms underlying coiled-coil system folding. The results presented in the thesis are of definite importance for possible applications (e.g. introduction of solvent explicitly into the scoring function) into protein folding, docking and rational design methods. The dissertation consists of four chapters: ● Chapter 1 contains an introduction to the topic of protein-protein interactions including basic concepts and an overview of the present state of research in the field. ● Chapter 2 focuses on the studies of the role of solvent in protein interfaces. ● Chapter 3 is devoted to the work on fluorinated amino acids in protein environments. ● Chapter 4 describes the study of coiled-coils folding properties. The experimental parts presented in Chapters 3 and 4 of this thesis have been performed by our collaborators at FU Berlin. Sections 2.1, 2.2, 3.1, 3.2 and Chapter 4 have been submitted/published in peer-reviewed international journals. Their organization follows a standard research article structure: Abstract, Introduction, Methodology, Results and discussion, and Conclusions. Section 3.3, though not published yet, is also organized in the same way. The literature references are summed up together at the end of the thesis to avoid redundancy within different chapters

How Atomic Level Interactions Drive Membrane Fusion: Insights From Molecular Dynamics Simulations

This project is focused on identifying the role of key players in the membrane fusion process at the atomic level with the use of molecular dynamics simulations. Membrane fusion of apposed bilayers is one of the most fundamental and frequently occurring biological phenomena in living organisms. It is an essential step in several cellular processes such as neuronal exocytosis, sperm fusion with oocytes and intracellular fusion of organelles to name a few. Membrane fusion is a frequent process in a living organism but is still not fully understood at the atomic level in terms of the role of various factors that play a crucial part in completion of membrane fusion. Two major factors that have been identified and studied experimentally are the protein Synaptotagmin and SNAREs. In addition, Ca2+ is known to play a crucial role in this process, however the exact mechanism of action is still unknown. Prime objective of this study is to understand these interactions and the role of Ca2 + in the process at the atomic level by carrying out molecular dynamics simulations. One of the primary calculations to perform is potential of mean force (PMF) between SYT and bilayer to analyze the effect of Ca2+ on their relative affinities. 1-octanol-water partition coefficient (log Kow) of a solute is a key parameter used in the prediction of a wide variety of complex phenomena such as drug availability and bioaccumulation potential of trace contaminants. Adaptive biasing force method is applied to calculate 1-octanol partition coefficients of n-alkanes and extended to other complex systems like ionic liquids, energetic materials and chemical warfare agents. Molecular dynamics simulations show that both domains of SYT-1, C2A and C2B, once calcium bound, insert into the lipid bilayer composed of anionic phospholipids. In contrast, no insertion is observed when the domains do not have bound calcium or when the bilayer is not charged negative. Electrostatic interactions play an important role in this insertion process. Effect of calcium binding to the C2A and C2B domain on the overall electrostatics of the protein was studied by generating the ESP maps. Negative potential on the Calcium binding pocket transforms into positive potential once calcium is attached to those sites. Interaction of this positive potential surface with the negatively charged bilayer acts as a driving force for protein insertion into the bilayer. In addition, adaptive biasing force method has emerged as a powerful tool for prediction of 1-octanol water partition coefficients and is successfully implemented and optimized for n-alkanes and extended to the systems of ionic liquids, energetic materials and chemical warfare agents for which 1-octanol water partition coefficient is either not known or is difficult to measure via experimental methods

Azucares y Proteínas: el papel de la dinámica en las interacciones moleculares

Tesis inédita de la Universidad Complutense de Madrid, Facultad de Farmacia, leída el 18/10/2016Glycans are among the most varied and complex molecules in biological systems. The different branches of the tree of life could be differentiated on the basis of the glycan composition of the own glycoconjugate molecules. However, how much we already know about glycans and glycoconjugate function and distribution is still an open question. Not so many years ago our knowledge about protein N-glycosylation was considerably scarce. In fact, while protein N-glycosylation was once believed to be limited to eukaryotes, it is now firmly established that this complex modification also occurs in bacteria and archaea. Consequently, in the past 10 years, the field of protein glycosylation has witnessed enormous strides in the discovery of new and unusual carbohydrates, in the elucidation of the enzymes involved in glycan assembly and processing, and in the understanding the biological impact that these glycan modifications have on the structure and function of target protein. The reason for this “late” discovery probably lies in the intrinsic structural complexity, heterogeneity and flexibility of glycans. As counterweight, numerous and exhaustive works in glycomics have demonstrated that it is due to their structural complexity, heterogeneity and flexibility why glycans have been selected as key intermediates for cell proliferation, differentiation, adhesion, infection, communication, etc. With this thesis we have tried to look inside into glycan structure, with the aim to reconcile their structural features at the atomic level with the reasons of their molecular flexibility at a more complex scale. When glycans are recognized by their receptors, their intrinsic flexibility and the plasticity of the whole system has enormous effects in the molecular recognition phenomenon. In fact, both partners involved in the intermolecular interaction could adapt their contact surface in a way that enhances enthalpy-based favourable intermolecular interactions. Alternatively or simultaneously, the glycan and the receptor could strategically keep internal molecular motions, even in the bound state, in a way that minimizes the entropy penalty to the binding event. As consequence, the role of enthalpic/entropic compensation is not easy to predict and even, to assess. Along this thesis we have explored these features, focusing our attention on sugar protein interactions, starting from the sugar flexibility at the monosaccharide level, passing then to the study of disaccharides, and later investigating the complex motions within a sugar receptor. Finally, CH/ intermolecular interactions, which essentially contribute to the stability of sugar-protein complexes, have also been discussed and a new strategy for their direct detection has been proposed...Los glicanos constituyen unos de los tipos de moléculas más variadas y complejas entre los sistemas biológicos. Las diferentes ramas del árbol de la vida se pueden distinguir en base a la composición de los glicanos de los propios glicoconjugados. Sin embargo, cuánto conocemos acerca de la función y distribución de los glicanos y glicoconjugados es una cuestión todavía abierta. Hace no demasiados años, nuestro conocimiento acerca de las glicoproteinas era considerablemente escaso. De hecho, durante mucho tiempo se creyó que la N-glicosilación de proteínas se daba sólo en los organismos eucariotas. Sin embargo, hoy está firmemente establecido que esta compleja modificación ocurre también en bacterias y arqueas. Por lo tanto, en los últimos 10 años, el campo de las glicoproteínas ha sido testigo de enormes avances en el descubrimiento de nuevos e inusuales carbohidratos, así como en la elucidación de las enzimas responsables de la construcción y procesamiento de glicanos y en la comprensión del impacto biológico que estas modificaciones tienen sobre la estructura y función de la proteína diana. La razón de este “retraso”, probablemente radica en la intrínseca complejidad estructural, heterogeneidad y flexibilidad de los glicanos. Como contrapeso, numerosos y exhaustivos trabajos en el campo de la glicómica han demostrado que es exactamente gracias a su complejidad estructural, heterogeneidad y flexibilidad el porqué los glicanos han sido seleccionados, entre otras biomoléculas, como intermedios claves para los procesos celulares de proliferación, diferenciación, adhesión, infección, comunicación, etc. En esta tesis, hemos intentado mirar profundamente en el interior de la estructura de los glicanos, con el objetivo de conciliar sus propiedades estructurales intrínsecas, a nivel atómico, con los motivos de su flexibilidad molecular. Cuando los glicanos interaccionan con sus receptores, esta flexibilidad se une a la plasticidad del sistema global para dar al lugar al proceso de reconocimiento molecular. De hecho, las dos partes que participan en la interacción intermolecular pueden adaptar su superficie de contacto de manera que se maximice la entalpia de unión. De manera alternativa o complementaria, el glicano y el receptor podrían mantener movimientos moleculares internos, incluso en el estado unido, de tal manera que se minimizase la penalización entrópica del proceso de unión. Como consecuencia de ello, el papel de la compensación entálpico/entrópica no es fácil de evaluar ni de medir...Fac. de FarmaciaTRUEunpu

Mechanistic insights into allosteric regulation of the A2A adenosine G protein-coupled receptor by physiological cations.

Cations play key roles in regulating G-protein-coupled receptors (GPCRs), although their mechanisms are poorly understood. Here, 19F NMR is used to delineate the effects of cations on functional states of the adenosine A2A GPCR. While Na+ reinforces an inactive ensemble and a partial-agonist stabilized state, Ca2+ and Mg2+ shift the equilibrium toward active states. Positive allosteric effects of divalent cations are more pronounced with agonist and a G-protein-derived peptide. In cell membranes, divalent cations enhance both the affinity and fraction of the high affinity agonist-bound state. Molecular dynamics simulations suggest high concentrations of divalent cations bridge specific extracellular acidic residues, bringing TM5 and TM6 together at the extracellular surface and allosterically driving open the G-protein-binding cleft as shown by rigidity-transmission allostery theory. An understanding of cation allostery should enable the design of allosteric agents and enhance our understanding of GPCR regulation in the cellular milieu