Dimerization Free Energy Research Articles

Evaluating Polyglutamine Peptide Monomers and Dimers with Enhanced Sampling MD Simulations

Huntington's disease is a neurodegenerative disease characterized by mutations causing polyglutamine (polyQ) repeats in proteins. Mutated proteins misfold, aggregate, and form amyloid-like fibrils in the neuron. Experimental techniques such as resonance Raman, circular dichroism, and ssNMR are used to analyze properties of polyQ systems, but lack the ability to monitor the molecular aggregation mechanism. In this work, polyQ peptide monomers and dimers are studied using molecular dynamics (MD) methods in order to better understand early stages of aggregation. We have characterized the monomeric conformational ensemble of D2Q10K2 peptides in aqueous and protein-globule environments. We also investigate dimerization properties of these peptides. Adaptive biasing force paired with MD is used to evaluate the dimerization free energies and conformations of D2Q10K2 peptides. Classical MD is used to simulate the favorable dimeric conformations in equilibrium, as to better understand the interactions and dynamics of these structures. These results will be presented and discussed.

Sequence-function relationships in folding upon binding.

Folding coupled to binding is ubiquitous in biology. Nevertheless, the relationship of sequence to function for protein segments that undergo coupled binding and folding remains to be determined. Specifically, it is not known if the well-established rules that govern protein folding and stability are relevant to ligand-linked folding transitions. Upon small ligand biotinoyl-5'-AMP (bio-5'-AMP) binding the Escherichia coli protein BirA undergoes a disorder-to-order transition that results in formation of a network of packed hydrophobic side chains. Ligand binding is also allosterically coupled to protein association, with bio-5'-AMP binding enhancing the dimerization free energy by -4.0 kcal/mol. Previous studies indicated that single alanine replacements in a three residue hydrophobic cluster that contributes to the larger network disrupt cluster formation, ligand binding, and allosteric activation of protein association. In this work, combined equilibrium and kinetic measurements of BirA variants with alanine substitutions in the entire hydrophobic network reveal large functional perturbations resulting from any single substitution and highly non-additive effects of multiple substitutions. These substitutions also disrupt ligand-linked folding. The combined results suggest that, analogous to protein folding, functional disorder-to-order linked to binding requires optimal packing of the relevant hydrophobic side chains that contribute to the transition. The potential for many combinations of residues to satisfy this requirement implies that, although functionally important, segments of homologous proteins that undergo folding linked to binding can exhibit sequence divergence.

Dimerization of FGFR3 in Living Cells

Receptor Tyrosine Kinases (RTKs) transduce biochemical signals via lateral dimerization in the membrane plane. Yet, our knowledge about the thermodynamics of RTK dimerization in cellular membranes is very limited. We are working to develop a FRET-based methodology to measure the dimerization free energies for RTKs in cells. We measure FRET with high spectral resolution, and we calculate the FRET efficiencies with high precision for each pixel of the cellular membrane. Here we present data for Fibroblast Growth Factor Receptor 3, an RTK that is critical for skeletal development. Since this receptor is activated by the ligand fgf1, we compare the FRET efficiencies with and without ligand. Consistent with the expectation that fgf1 stabilizes the FGFR3 dimer, we see an increase in the FRET signal.

Sequence-Function Relationships in Allostery Mediated by Disorder-To-Order Transitions

Allostery functions in many biological systems including signal transduction, transcription regulation, and metabolism. Although disorder-to-order transitions contribute to numerous allosteric processes, the relationship of the functional allosteric response to the sequences that undergo these transitions is not known. The Escherichia coli biotin repressor, BirA, is an allosteric transcription regulatory protein that undergoes ligand-induced loop folding upon corepressor, bio-5′-AMP, binding. Ligand binding and dimerization are coupled processes with bio-5′-AMP binding enhancing the dimerization free energy by −4.0 kcal/mole. Previous work has demonstrated that adenylate binding is coupled to folding of a loop in which any single alanine replacement disrupts effector binding, loop folding and functional coupling. In this work, coupling between residues that contribute to the disorder-to-order transition was investigated by studying variant proteins with several combinations of alanine substitutions. Combined kinetic and equilibrium measurements reveal non-additive effects of multiple amino acid substitutions for all BirA functions. The results also indicate that specific combinations of alanine substitutions lead to reversion of the allosteric response toward that observed for the wild type protein. In combination the results suggest that, analogous to protein folding, full function in disorder-to-order transitions requires appropriate packing of the relevant side chains.

Dimerization hot spots in the structure of human Hsp90

Molecular dynamics and dimerization free energy analyses performed on the human Hsp90 dimer highlight dimerization hot spots and potential allosteric binding sites.

Assessing Polyglutamine Conformation and Aggregation with Molecular Dynamics Techniques

Huntington's disease is one of nine neurodegenerative diseases characterized by gene mutations causing polyglutamine (polyQ) repeats in various proteins. Mutated proteins misfold, aggregate, and form amyloid-like fibrils in the neuron. As of now, the aggregation mechanism of these polyglutamine proteins is not well understood. Experimental techniques such as resonance Raman, circular dichroism, and ssNMR are used to analyze properties of polyglutamine solutions. Computational analysis is used in concert with experiment to allow investigation on the molecular level. In this work, short polyQ peptides are studied using molecular dynamics (MD) methods in order to better understand the mechanics of their aggregation. In prior work, we characterized the monomeric conformational ensemble of D2Q10K2 peptides. Our next step is to investigate dimerization properties of these peptides. Adaptive biasing force paired with MD is used to evaluate the dimerization free energies and conformations of D2Q10K2 peptides. On a larger scale, classical MD is used to evaluate the properties of multiple aggregate conformations, including facially stacked β-sheet and β-hairpin sheet systems. Ψ angle probability distributions will be generated from the resulting aggregate trajectories for comparison with experimental distributions. Results from both projects will be presented.

A Large Solvent Isotope Effect on Protein Association Thermodynamics

Solvent reorganization can contribute significantly to the energetics of protein-protein interactions. However, our knowledge of the magnitude of the energetic contribution is limited, in part, by a dearth of quantitative experimental measurements. The biotin repressor forms a homodimer as a prerequisite to DNA binding to repress transcription initiation. At 20 °C, the dimerization reaction, which is thermodynamically coupled to binding of a small ligand, bio-5'-AMP, is characterized by a Gibbs free energy of -7 kcal/mol. This modest net dimerization free energy reflects underlying, very large opposing enthalpic and entropic driving forces of 41 ± 3 and -48 ± 3 kcal/mol, respectively. The thermodynamics have been interpreted as indicating coupling of solvent release to dimerization. In this work, this interpretation has been investigated by measuring the effect of replacing H2O with D2O on the dimerization thermodynamics. Sedimentation equilibrium measurements performed at 20 °C reveal a solvent isotope effect of -1.5 kcal/mol on the Gibbs free energy of dimerization. Analysis of the temperature dependence of the reaction in D2O indicates enthalpic and entropic contributions of 28 and -37 kcal/mol, respectively, considerably smaller than the values measured in H2O. These large solvent isotope perturbations to the thermodynamics are consistent with a significant contribution of solvent release to the dimerization reaction.

Role of Dimerization Efficiency of Transmembrane Domains in Activation of Fibroblast Growth Factor Receptor 3

Mutations in transmembrane (TM) domains of receptor tyrosine kinases are shown to cause a number of inherited diseases and cancer development. Here, we use a combined molecular modeling approach to understand molecular mechanism of effect of G380R and A391E mutations on dimerization of TM domains of human fibroblast growth factor receptor 3 (FGFR3). According to results of Monte Carlo conformational search in the implicit membrane and further molecular dynamics simulations, TM dimer of this receptor is able to form a number of various conformations, which differ significantly by the free energy of association in a full-atom model bilayer. The aforementioned mutations affect dimerization efficiency of TM segments and lead to repopulation of conformational ensemble for the dimer. Particularly, both mutations do not change the dimerization free energy of the predominant (putative "non-active") symmetric conformation of TM dimer, while affect dimerization efficiency of its asymmetric ("intermediate") and alternative symmetric (putative "active") models. Results of our simulations provide novel atomistic prospective of the role of G380 and A391E mutations in dimerization of TM domains of FGFR3 and their consecutive contributions to the activation pathway of the receptor.

On the Relative Stability of Dimeric Interfaces of the Mu-Opioid Receptor Inferred from Recent Crystallographic Studies

The mu-opioid (MOP) receptor is the predominant target for most clinically used analgesics. In response to demonstrated correlations between opioid dependence and the MOP receptor, researchers have focused on alternative targets. Opioid receptor oligomers are among those that have been suggested to mediate analgesia without the common opioid-related adverse effects. Thus, obtaining a molecular-level understanding of the nature of receptor-receptor interactions in the membrane, either within, or between receptor subtypes, can both create new opportunities for drug discovery, and address the role of oligomerization in receptor function. The recent MOP receptor crystal structure has inspired hypotheses of dimerization contacts, specifically: a closely packed interface involving transmembrane (TM) helices TM5 and TM6, and a less compact one involving TM1, TM2, and helix 8 (H8). These interfaces exhibit similar arrangements to those found in crystals of the chemokine receptor CXCR4 and the kappa-opioid receptor, respectively. While it is tempting to speculate that the tighter TM5/TM6 arrangement has physiological relevance, additional studies are necessary to a) understand the relative dimer stability at TM5/TM6, TM1/TM2/H8, and other interfaces in the membrane, b) evaluate the contribution of the engineered T4 lysozyme (T4L) to the TM5/TM6 association, and c) investigate possible variability across different receptors. To begin to address these questions we have performed umbrella sampling molecular dynamics simulations of coarse-grained representations of the MOP receptor interacting at the TM5/TM6 (with and without T4L) or TM1/TM2/H8 interfaces in an explicit lipid-water environment. We have derived relative estimates of the dimerization free energy at the two specific interfaces and from these we suggest relative dimer lifetimes and dimeric fractions in a lipid mimetic environment. This information can help design future experiments aimed at understanding the role of dimerization in receptor function.

Role of F− in the hydrolysis–condensation mechanisms of silicon alkoxide Si(OCH3)4: a DFT investigation

The hydrolysis and condensation mechanisms of tetramethoxysilane aided by F− were investigated with Gaussian03 program package. The coordination of F− to tetramethoxysilane and the first-order hydrolysis of the fluorotetramethoxysilane anion were studied extensively with both CPCM full optimization and CPCM single-point energy (SPE) calculations, and the single-point energies agree well with those obtained from the full optimization with CPCM solvation model. The coordination of F− decreases the Mulliken charge on the Si atom and the energy gap between the HOMO and LUMO, and alters the shape of the frontier orbitals. Our calculations show that entropic effects elevate potential energy surface (PES) profiles distinctly, but have a minor influence on the free energy barriers. With the aid of F−, the hydrolysis barriers of fluorotetramethoxysilane anion and the dimerization free energy barriers before entropic corrections decrease from 104, 106, 109, 109 and 136, 104, 99, 94 to kJ mol−1 to 84.2, 88.5, 77.2, 81.9 and 82.2, 80.0, 87.7, 88.7 kJ mol−1, respectively, compared with the neutral hydrolysis and condensation barriers. These barriers are much lower than the corresponding neutral SN2 ones, but only slightly higher than the SN1 ones. The role of nucleophile F− is similar to HO−.

Improved Parameters for the Martini Coarse-Grained Protein Force Field.

The Martini coarse-grained force field has been successfully used for simulating a wide range of (bio)molecular systems. Recent progress in our ability to test the model against fully atomistic force fields, however, has revealed some shortcomings. Most notable, phenylalanine and proline were too hydrophobic, and dimers formed by polar residues in apolar solvents did not bind strongly enough. Here, we reparametrize these residues either through reassignment of particle types or by introducing embedded charges. The new parameters are tested with respect to partitioning across a lipid bilayer, membrane binding of Wimley-White peptides, and dimerization free energy in solvents of different polarity. In addition, we improve some of the bonded terms in the Martini protein force field that lead to a more realistic length of α-helices and to improved numerical stability for polyalanine and glycine repeats. The new parameter set is denoted Martini version 2.2.

Assessing the Relative Stability of Dimer Interfaces in G Protein-Coupled Receptors

Considerable evidence has accumulated in recent years suggesting that G protein-coupled receptors (GPCRs) associate in the plasma membrane to form homo- and/or heteromers. Nevertheless, the stoichiometry, fraction and lifetime of such receptor complexes in living cells remain topics of intense debate. Motivated by experimental data suggesting differing stabilities for homomers of the cognate human β1- and β2-adrenergic receptors, we have carried out approximately 160 microseconds of biased molecular dynamics simulations to calculate the dimerization free energy of crystal structure-based models of these receptors, interacting at two interfaces that have often been implicated in GPCR association under physiological conditions. Specifically, results are presented for simulations of coarse-grained (MARTINI-based) and atomistic representations of each receptor, in homodimeric configurations with either transmembrane helices TM1/H8 or TM4/3 at the interface, in an explicit lipid bilayer. Our results support a definite contribution to the relative stability of GPCR dimers from both interface sequence and configuration. We conclude that β1- and β2-adrenergic receptor homodimers with TM1/H8 at the interface are more stable than those involving TM4/3, and that this might be reconciled with experimental studies by considering a model of oligomerization in which more stable TM1 homodimers diffuse through the membrane, transiently interacting with other protomers at interfaces involving other TM helices.

How Do α-Cyclodextrins Self-Organize on a Polymer Chain?

The relative conformation of the mobile cyclic molecules of a polyrotaxane has been analyzed quantitatively by means of both molecular dynamics and Monte Carlo simulations. Here, the polyrotaxane is formed by several α-cyclodextrins (α-CDs) threaded onto a poly(ethylene glycol) (PEG) chain. The dimerization free energies for three possible spatial arrangements of two consecutive α-CDs, viz., head to head (HH), head to tail (HT), and tail to tail (TT), were determined. The computed dimerization free energies were then introduced into the theoretical framework of a lattice model to predict the percentage of HH and TT motifs in all possible arrangements, employing Monte Carlo simulations. Our results show that this percentage fluctuates when the number of CDs is less than eight and rapidly tends toward 73% when the latter is greater than eight. This theoretical estimate, which is dominated by the dimerization free energy, agrees well with experiments. Deconvolution of the free-energy profiles indicates that ...

Glucocorticoid Receptor–Promoter Interactions: Energetic Dissection Suggests a Framework for the Specificity of Steroid Receptor-Mediated Gene Regulation

The glucocorticoid receptor (GR) is a member of the steroid receptor family of ligand-activated transcription factors. A number of studies have shown that steroid receptors regulate distinct but overlapping sets of genes; however, the molecular basis for such specificity remains unclear. Previous work from our laboratory has demonstrated that under identical solution conditions, three other steroid receptors [the progesterone receptor A isoform (PR-A), the progesterone receptor B isoform (PR-B), and estrogen receptor α (ER-α)] differentially partition their self-association and promoter binding energetics. For example, PR-A and PR-B generate similar dimerization free energies but differ significantly in their extents of intersite cooperativity. Conversely, ER-α maintains an intersite cooperativity most comparable to that of PR-A yet dimerizes with an affinity orders of magnitude greater than that of either of the PR isoforms. We have speculated that these differences serve to generate receptor-specific promoter occupancies, and thus receptor-specific gene regulation. Noting that GR regulates a unique subset of genes relative to the other receptors, we hypothesized that the receptor should maintain a unique set of interaction energetics. We rigorously determined the self-association and promoter binding energetics of full-length, human GR under conditions identical to those used in our earlier studies. We find that unlike all other receptors, GR shows no evidence of reversible self-association. Moreover, GR assembles with strong intersite cooperativity comparable to that seen only for PR-B. Finally, simulations show that such partitioning of interaction energetics allows for receptor-specific promoter occupancies, even under conditions where multiple receptors are competing for binding at identical sites.

Dimerization of Amino Acid Side Chains: Lessons from the Comparison of Different Force Fields.

The interactions between amino acid side chains govern protein secondary, tertiary, and quaternary structure formation. For molecular modeling approaches to be able to realistically describe these phenomena, the underlying force fields have to represent these interactions as accurately as possible. Here, we compare the side chain-side chain interactions for a number of commonly used force fields, namely the all-atom OPLS, the united-atom GROMOS, and the coarse-grain MARTINI force field. We do so by calculating the dimerization free energies between selected pairs of side chains and structural characterization of their binding modes. To mimic both polar and nonpolar environments, the simulations are performed in water, n-octanol, and decane. In general, reasonable correlations are found between all three force fields, with deviations on the order of 1 kT in aqueous solvent. In apolar solvent, however, significantly larger differences are found, especially for charged amino acid pairs between the OPLS and GROMOS force fields, and for polar interactions in the MARTINI force field in comparison to the higher resolution models. Interestingly, even in cases where the dimerization free energies are similar, the binding mode may differ substantially between the force fields. This was found to be especially the case for aromatic residues. In addition to the inter-force-field comparison, we compared the various force fields to a knowledge-based potential. The two independent approaches show good correlation in aqueous solvent with an exception of aromatic residues for which the interaction strength is lower in the knowledge-based potentials.

Influence of the Chemical Functionalities of a Molecularly Imprinted Conducting Polymer on Its Sensing Properties: Electrochemical Measurements and Semiempirical DFT Calculations

Starting from thiophene-based functional monomers (FM), namely, TMA, TAA, TMeOH, EDOT, and Th, bonded to atrazine (ATZ) target molecules into FM/ATZ prepolymerization dimers in acetonitrile solutions, differently functionalized molecularly imprinted conducting polymers (FM-MICP) are electrosynthesized and then washed and used as sensitive layers for ATZ recognition. Sensitivity of these layers toward ATZ, which is quantified by cyclic voltammetric measurements, decreases in the following order of functional monomers: TMA, TAA, TMeOH, EDOT, and Th. Absolute values of the FM-ATZ dimerization free energies are calculated with the help of DFT/PCM calculations and of an empirical correction of the entropy effects, using a modified Wertz formula. A strong correlation is found between FM-MICP sensitivity and the amount of FM/ATZ prepolymerization complexes.

Thermodynamic Dissection of Estrogen Receptor–Promoter Interactions Reveals That Steroid Receptors Differentially Partition Their Self-Association and Promoter Binding Energetics

Steroid receptors define a family of ligand-activated transcription factors. Recent work has demonstrated that the receptors regulate distinct but overlapping gene networks, yet the mechanisms by which they do so remain unclear. We previously determined the microscopic binding energetics for progesterone receptor (PR) isoform assembly at promoters containing multiple response elements. We found that the two isoforms (PR-A and PR-B) share nearly identical dimerization and intrinsic DNA binding free energies but maintain large differences in cooperative free energy. Moreover, cooperativity can be modulated by monovalent ion binding and promoter layout, suggesting that differences in cooperativity might control isoform-specific promoter occupancy and thus receptor function. To determine whether cooperative binding energetics are common to other members of the steroid receptor family, we dissected the thermodynamics of estrogen receptor-α (ER-α):promoter interactions. We find that the ER-α intrinsic DNA binding free energy is identical to that of the PR isoforms. This was expected, noting that receptor DNA binding domains are highly conserved. Unexpectedly, ER-α generates negligible cooperativity-orders of magnitude less than predicted based on our studies of the PR isoforms. However, analysis of the cooperativity term suggests that it reflects a balance between highly favorable cooperative stabilization and unfavorable promoter bending. Moreover, ER-α cooperative free energy is compensated for by a large increase in dimerization free energy. Collectively, the results demonstrate that steroid receptors differentially partition not only cooperative energetics but also dimerization energetics. We speculate that this ability serves as a framework for regulating receptor-specific promoter occupancy and thus receptor-specific gene regulation.

Insight into the Thermodynamics and Equilibrium Kinetics of the Interaction between Transmembrane α-Helices in the Membrane Domain of ErbB4

Interactions between the transmembrane (TM) α-helices in the membrane domains (MD) of integral membrane proteins are believed to determine their spatial structure and functionality. Nevertheless, the basic principles underlying such interactions still need to be elucidated. In the present work, the interaction between the TM segments in two-helical MD of the receptor tyrosine kinase ErbB4 was investigated by means of solution NMR spectroscopy in lipidic bicelles. According to the obtained data, the α-helical TM domains of the receptor associate via the double motif A655GxxGG660, forming the parallel dimer, stabilized by the polar contacts. The slow character of dimer-monomer transition of the ErbB4 TM domains permitted to access the thermodynamics and equilibrium kinetics of the dimerization. Lipidic bicelles appeared to be an ideal solvent in terms of dimer-monomer equilibrium of the ErbB4 TM domains, which allowed to measure the free energy of their dimerization equal to −1.4 kcal/mol. Noteworthy, the dimerization constant began to increase dramatically when more than one peptide on average were induced to reside in one bicelle (“saturated bicelles”). Enthalpy, entropy and heat capacity of the dimerization were obtained from the temperature dependence of the dimerization constant in “saturated bicelles”. As was shown, the dimerization of TM domains of ErbB4 is an endothermic process, going with the substantial growth of entropy and heat capacity of the system, suggesting the important role of lipids in the TM helix-helix interactions. The temperature dependence of linewidths of NMR signals was interpreted in terms of the reaction rates and the transition state theory to derive the free energy, entropy and enthalpy of the transition state and to estimate the contribution from the lipid-protein and protein-protein interactions into the free energy of dimerization.

Thermodynamic Dissection of Estrogen Receptor-Promoter Interactions Reveals that Steroid Receptor Family Members Differentially Partition their Binding Energetics

The quantitative principles responsible for steroid receptor-specific gene regulation are poorly understood. This is in part because most studies have measured only apparent binding affinities or used biochemical rather than biophysical approaches. We previously dissected the thermodynamics of human progesterone receptor (PR) isoform assembly at multisite promoter sequences. We found that the two isoforms maintain surprisingly weak dimerization energetics; however, the receptors can achieve full promoter occupancy via strong cooperative interactions. As a step towards assessing whether cooperative binding energetics are a common feature for all steroid receptors, we dissected the thermodynamics of human estrogen receptor-∗ (ER-∗):promoter interactions under conditions identical to our work on PR. Analytical ultracentrifugation and quantitative footprint titrations demonstrate that ER-∗ and PR dimers maintain similar intrinsic binding affinities toward their respective response elements. This is to be expected noting the near identical structures of the ER and PR DNA binding domains. Unexpectedly, however, ER-∗ exhibits negligible cooperativity - orders of magnitude less than predicted based on our studies of PR. This raises the question of how ER-∗ generates full occupancy (and thus full function) at multisite promoters. Our results reveal that the decrease in ER-∗ cooperative free energy is exactly compensated for via an increase in dimerization free energy. Thus homologous transcription factors partition their promoter binding energetics differentially. We speculate that this serves as a mechanism to receptor-specific gene regulation: differences in assembly state and cooperative stabilization allow for preferential occupancy as a function of response element layout.

A Simple Theory for Entropic Interaction Induced between Large Spheres in a Binary Mixture of Small and Medium Spheres

We develop a simple theory for the entropic interaction between large spheres immersed in a binary mixture of small and medium spheres. In the context of the Asakura–Oosawa-type theory, the densities of small and medium spheres were assumed to be uniform except within the excluded volumes generated by the large spheres. In this study, two essential terms are newly formulated as correction terms for the uniform density approximation employed in the theory. The first part represents the entropic gain arising from the holding of a medium sphere which accompanies an increase in the total volume available to the translational displacement of small spheres and the other medium spheres. The second one accounts for the entropic loss caused by the restriction of translational freedom of the medium sphere held. It is important to incorporate both of the two parts in the theory. The resulting theory, which gives us semi-quantitatively correct results for the dimerization free energy of large spheres (the difference from the result calculated by the integral equation theory is only ∼20% at usual total packing fractions), enables us to provide a new mechanism of the association of macromolecules arising from the addition of crowding agents.