Dimerization Free Energy Research Articles

Multiple Hydrogen-Bonded Complexes Based on 2-Ureido-4[1H]-pyrimidinone: A Theoretical Study

In the present work, the electronic structures and properties of a series of 2-ureido-4[1H]-pyrimidinone(UPy)-based monomers and dimers in various environments (vacuum, chloroform, and water) are studied by density functional theoretical methods. Most dimers prefer to form a DDAA-AADD (D, H-bond donor; A, H-bond acceptor) array in both vacuum and solvents. Topological analysis proved that intramolecular and intermolecular hydrogen bonds coexist in the dimers. Frequency and NBO calculations show that all the hydrogen bonds exhibit an obvious red shift in their stretching vibrational frequencies. Larger substituents at position 6 of the pyrimidinone ring with stronger electron-donating ability favor the total binding energy and free energy of dimerization. Calculations on the solvent effect show that dimerization is discouraged by the stronger polarity of the solvent. Further computations show that Dimer-1 may be formed in chloroform, but water molecules may interact with the donor or acceptor sites and hence disrupt the hydrogen bonds of Dimer-1.

Determining equilibrium constants for dimerization reactions from molecular dynamics simulations

With today's available computer power, free energy calculations from equilibrium molecular dynamics simulations "via counting" become feasible for an increasing number of reactions. An example is the dimerization reaction of transmembrane alpha-helices. If an extended simulation of the two helices covers sufficiently many dimerization and dissociation events, their binding free energy is readily derived from the fraction of time during which the two helices are observed in dimeric form. Exactly how the correct value for the free energy is to be calculated, however, is unclear, and indeed several different and contradictory approaches have been used. In particular, results obtained via Boltzmann statistics differ from those determined via the law of mass action. Here, we develop a theory that resolves this discrepancy. We show that for simulation systems containing two molecules, the dimerization free energy is given by a formula of the form ΔG ∝ ln(P(1) /P(0) ). Our theory is also applicable to high concentrations that typically have to be used in molecular dynamics simulations to keep the simulation system small, where the textbook dilute approximations fail. It also covers simulations with an arbitrary number of monomers and dimers and provides rigorous error estimates. Comparison with test simulations of a simple Lennard Jones system with various particle numbers as well as with reference free energy values obtained from radial distribution functions show full agreement for both binding free energies and dimerization statistics.

First Principles Calculations of Atomic Nickel Redox Potentials and Dimerization Free Energies: A Study of Metal Nanoparticle Growth.

The redox potentials and dimerization free energies of transient transition metal cations in water shed light on the reactivity of species with unusual charge states and are particularly pertinent to understanding the mechanism and feasibility of radiolysis-assisted metal nanoparticle growth from salt solutions. A combination of quasi-chemical theory and ab initio molecular dynamics thermodynamic integration methods are applied to calculate these properties for nickel. The reduction potential for Ni(2+) (aq) is predicted to be between -1.05 to -1.28 V, which is substantially lower than previous estimates. This suggests that Ni(2+) reduction may possibly occur in the presence of organic radical anion electron scavengers and hydrogen atoms, not just hydrated electrons. In contrast, Ni(+) is found to be stable against disproportionation. The formation of dimers Ni2 and Ni2(+) from Ni and Ni(+) are predicted to be favorable in water.

Dimerization free energy of vancomycin‐group antibiotics and the cooperative effect: A density functional approach

AbstractThe cooperative effects between the binding of model bacterial cell wall termini and the dimer forms of the glycopeptide antibiotic vancomycin and chloro‐eremomycin were investigated with conventional and divide‐and‐conquer (DC) ab initio quantum chemistry simulations. This feature is potentially important for drug design and controlling molecular recognition phenomena in these and other biochemical systems. In terms of the calculation, the vibrational contributions to the dimerization free energies of both molecules found to be the most costly and necessitated the use of the DC method. We specifically focused on calculating the relative strength of the cooperative effect for the two antibiotics. In good agreement with experiments, we find that the cooperative effect is stronger for chloro‐eremomycin by about 1.39 kcal/mol. © 2010 Wiley Periodicals, Inc. Int J Quantum Chem, 2010

Physical Basis behind Achondroplasia, the Most Common Form of Human Dwarfism

Fibroblast growth factor receptor 3 (FGFR3) is a receptor tyrosine kinase that plays an important role in long bone development. The G380R mutation in FGFR3 transmembrane domain is known as the genetic cause for achondroplasia, the most common form of human dwarfism. Despite many studies, there is no consensus about the exact mechanism underlying the pathology. To gain further understanding into the physical basis behind the disorder, here we measure the activation of wild-type and mutant FGFR3 in mammalian cells using Western blots, and we analyze the activation within the frame of a physical-chemical model describing dimerization, ligand binding, and phosphorylation probabilities within the dimers. The data analysis presented here suggests that the mutation does not increase FGFR3 dimerization, as proposed previously. Instead, FGFR3 activity in achondroplasia is increased due to increased probability for phosphorylation of the unliganded mutant dimers. This finding has implications for the design of targeted molecular treatments for achondroplasia.

Insights into the Recognition and Association of Transmembrane α-Helices. The Free Energy of α-Helix Dimerization in Glycophorin A

Insights into the Recognition and Association of Transmembrane α-Helices. The Free Energy of α-Helix Dimerization in Glycophorin A

A Molecular Dynamics Study of Amyloid-β (1-42) Peptide Dimer Formation on the Surface of Phospholipid Bilayers

The Amyloid-β (Aβ) peptide is an integral aggregate species in the progression and neurotoxicity of Alzheimer's disease. While Aβ fibrils were historically considered the toxic species in Alzheimer's disease, recent evidence has shifted the focus towards oligomers as the most dangerous aggregate structure for neurons. In this aggregation process, the conversion of monomeric Aβ into a dimer constitutes the first step in oligomer formation. Further work has shown that cell membranes may play a substantial role in promoting aggregation through facilitating the protein-protein interactions that drive aggregation. We have used extensive replica exchange molecular dynamics simulations to demonstrate that monomeric Aβ does not adopt stable secondary structure over timescales that have allowed for significant structure formation in solution. Further, to characterize dimer formation on the surface of a model lipid bilayer, we have used a thermodynamic cycle to indirectly calculate free energies of dimerization on the bilayer surface. Use of a thermodynamic cycle helps to decrease bias due to initial conditions that would occur through directly calculating a dimerization free energy. We have calculated the free energies of dimerization for a preformed dimer containing a single antiparallel β-sheet or a pair of β-hairpin monomers. While these structures are representative of predicted fibril structures, comparison of dimerization free energies provides insight into the effect of the bilayer on the dimerization process. We have found that the bilayer does affect dimerization free energy depending on the dimer structure and bilayer surface charge. Our work demonstrates that a lipid bilayer is able to substantially hinder Aβ monomer structure formation and influence Aβ dimer formation.

Quantitative Measurements of Receptor Interactions in Mammalian Cells: Implications for Human Pathologies

Receptor Tyrosine kinases (RTKs) are family of single-pass cell membrane receptors with extracellular ligand-binding domains and intracellular kinase domains, which conduct biochemical signals via lateral dimerization in the plasma membrane. Mutations in the transmembrane (TM) domains of these receptors are known to promote unregulated signaling. An example is the Ala391Glu mutation in the TM domain of FGFR3, which leads to pathologies such as Crouzon syndrome and bladder cancer. This mutation has been shown to stabilize the isolated TM domain dimers in lipid membranes, but it is not known if it stabilizes the full length FGFR3 receptor dimers in the plasma membrane of mammalian cells. To address the effect of the mutation in mammalian cells, we have determined free energies of dimerization for the wild type and mutant FGFR3 in mammalian (HEK293T and CHO) plasma membranes using the QI Förster resonance energy transfer (FRET) technique [Li et al.,2008]. The measured change in the dimerization free energy due to the Ala391Glu mutation is −1.2 kcal/mol, consistent with previous reports of hydrogen bond strength in proteins, as well as results for the isolated FGFR3 TM domains. Thus, we have shown that the mutation stabilizes the full length FGFR3 dimers in mammalian cells. We propose that this dimer stabilization is the major cause for FGFR3 overactivation and human pathologies. Li E,Placone J,Merzlyakov M, Hristova K (2008) Quantitative measurements of protein interactions in a crowded cellular environment.Anal Chem 80:5976-5985.

A DFT investigation of dissociation of nitrous acid, transformation of its monomeric isomers, their dimeric isomers, and dimerization

AbstractThe structure optimization of the monomeric isomers of the nitrous acid HONO, their cyclic dimers (HONO)2, and transition states of their transformations has been carried out using the density functional theory calculations at the B3LYP/6‐311++G(d,p) and B3LYP/aug‐cc‐pVQZ levels of theory. Three monomeric isomers (c, t, and z isomers) and six dimeric isomers (cc, ct, cz, tt, zz, and tz isomers) were found. Dissociation of the most stable of the HONO isomer (t isomer) was investigated and its rate constant was obtained at the UB3LYP/aug‐cc‐pVQZ level. Energetics, thermodynamic properties, rate, and equilibrium constants of the monomeric and dimeric isomerizations of the nitrous acid were obtained. Dimerization energies, enthalpies, and free energies of the dimeric isomers, (HONO)2 were also obtained. © 2009 Wiley Periodicals, Inc. Int J Quantum Chem, 2010

Lipids Enhance Apolipoprotein C-II-Derived Amyloidogenic Peptide Oligomerization but Inhibit Fibril Formation

We investigated the effect of submicellar lipids on amyloid fibril formation. Thioflavin T fluorescence studies showed that submicellar levels of the short-chain phospholipids, dipentanoylphosphatidylcholine and dihexanoylphosphatidylcholine, strongly inhibited amyloid fibril formation by an 11-residue peptide derived from human apolipoprotein C-II (apoC-II(60-70)). In contrast, sedimentation equilibrium analysis of these peptide-lipid mixtures indicated the presence of soluble oligomeric complexes. To acquire insight into the atomic level influences of these lipids on the initial stages of aggregation of the peptide, we performed molecular dynamics (MD) simulations coupled with umbrella sampling to determine dimerization free energies of a number of beta-stranded and random coil dimer complexes, both in the presence and absence of lipids. The simulations indicate that, in contrast to their inhibitory effects on fibril formation, short-chain phospholipids promote the formation and stabilization of dimers by enhancing intersubunit hydrophobic interactions. On the basis of these experimental and computational results, we propose that peptide-bound lipids can inhibit amyloid fibril formation by trapping of dimers and other oligomeric species in diverse nonfibril forming conformations, reducing their likelihood of acquiring subunit conformations prone to fibril nucleation and growth. In light of the demonstrated cytotoxicity of amyloid peptide oligomers, our results suggest that, by enhancing the stability of oligomeric peptide species, the presence of solvated lipids may contribute to the cytotoxicity of fibrillogenic proteins and peptides.

Association of Helical β-Peptides and their Aggregation Behavior from the Potential of Mean Force in Explicit Solvent

Helical β-peptides have been shown to fold into well-defined structures. In aqueous solution, some β-peptides self-assemble into nanoscale fibers, aggregates, and liquid crystalline phases. Molecular simulations, at an atomistic level, are used to examine, in a systematic manner, the interactions between distinct β-peptide molecules. The relationship between side-chain chemistry (and position along the backbone) and, in particular, aggregation behaviors, is assessed by calculating the potential of mean force or dimerization free energy of two peptides in explicit water. The free energy profiles as a function of separation for helical, amphiphilic β-peptides are consistent with experimental observations, and help explain the origins of aggregate or fiber formation in solution. Close examination of the energetic and entropic contributions to the free energy reveals that, depending on the position of certain side groups along the molecule, the tendency of two peptides to aggregate can be driven by entropy or by energy, respectively. In contrast to findings from previous works that employed a coarse representation of the solvent, it is shown that water-peptide interactions play key roles in the association behavior of β-peptides.

ChemInform Abstract: Homodimerization of Neurotensin 1 Receptor Involves Helices 1, 2, and 4: Insights from Quaternary Structure Predictions and Dimerization Free Energy Estimations.

AbstractChemInform is a weekly Abstracting Service, delivering concise information at a glance that was extracted from about 200 leading journals. To access a ChemInform Abstract of an article which was published elsewhere, please select a “Full Text” option. The original article is trackable via the “References” option.

Outer Membrane Phospholipase A Dimer Stability Does Not Correlate to Occluded Surface Area

Despite the key roles of oligomeric membrane proteins (MPs) in many known cellular pathways, the principles governing their oligomer stability are not well-understood. Previous work with the alpha-helical MPs bacteriorhodopsin (bR) and glycophorin A (GpA) shows that lost buried surface area linearly correlated with perturbations in protein stability. Although this is a significant discovery, the predictive power of this correlation is limited by the data. Because both bR and GpA have alpha-helical secondary structural motifs, it is unclear whether this correlation would be observed for MPs with a beta-barrel motif. We addressed this question by measuring the thermodynamic consequences of interfacial amino acid changes at the dimer interface of beta-barrel MP outer membrane phospholipase A (OMPLA). We created sequence variants to reduce the contact surface area of the OMPLA dimer interface by introducing single-alanine substitutions and used sedimentation equilibrium analytical ultracentrifugation to determine the dimerization free energies for these variants. The integrity of each variant was verified by two functional assays: specific activity and resistance to thermal denaturation, which showed that structural changes were restricted to the local environment. Using this information, we calculated the anticipated packing defects due to side chain deletion and compared this to the free energy perturbations for each residue. Contrary to the findings with bR and GpA, our study found no correlation between the contact surface area lost and the perturbations to OMPLA dimer stability. We conclude that van der Waals packing may not be a strong predictor of stability for all membrane proteins.

Homodimerization of Neurotensin 1 Receptor Involves Helices 1, 2, and 4: Insights from Quaternary Structure Predictions and Dimerization Free Energy Estimations

A computational approach based upon rigid-body docking, ad hoc filtering, and cluster analysis has been combined with a protocol for dimerization free energy estimations to predict likely interfaces in the neurotensin 1 receptor (NTS1) homodimers. The results of this study suggest that the likely intermonomer interfaces compatible with in vitro binding affinity constants essentially involve helices 1, 2, and 4 and do not include disulfide bridges. The correlative model initially developed on Glycophorin A and herein extended to a G protein-Coupled Receptor (GPCR) appears to be a useful tool for estimating the association free energies of transmembrane proteins independent of the size and shape of the interface. In the desirable future cases, in which in vitro intermonomer binding affinities will be available for other GPCRs, such a correlative model will work as an additional criterion for helping in the selection of the most likely dimers.

Basis Set and Electron Correlation Effects on Lithium Carbenoid Dimerization Energies

Abstract A systematic investigation was performed to determine which levels of theory are required to obtain accurate geometries and dimerization energies for lithium carbenoids. Dimerization free energies of six lithium carbenoids were calculated in the gas phase at the B3LYP and MP2 levels with several different basis sets and at the CCSD(T) level with the aug-cc-pvdz basis set. It was found that the calculated thermal corrections to the dimerization free energy, obtained from frequency calculation, were relatively independent of the basis set for basis sets larger than 6-31+G(d) and that smaller basis sets often generated qualitatively correct results. The dimerization free energies were usually overestimated by small basis sets compared to those using polarization and diffuse functions, whereas only modest gains were obtained by using basis sets larger than 6-31+G(d). The B3LYP DFT method generated geometries consistent with the MP2 and CCSD methods for halomethyllithium carbenoids, but not always for 1-halovinyllithium carbenoids. The B3LYP calculations also severely underestimated the dimerization free energies for the halovinyllithium carbenoids, compared to the MP2, and coupled cluster methods.

Modeling lithium dialkylamides in ethereal solvents: A test of the microsolvation model

The dimerization energies of lithium diisopropylamide (LDA), lithium tetramethylpiperidide (LiTMP), and lithium hexamethyldisilazide (LiHMDS) were calculated with the hybrid B3LYP DFT method. The effects of ethereal solvents were modeled using explicit dimethyl ether or THF ligands. The trends in calculated dimerization free energies were in good agreement with the observed aggregation states of these compounds in THF. Substitution of the THF ligands by the computationally less demanding dimethyl ether introduced significant error in the calculated dimerization free energies. This solvent model also generated structures and energies of lithium diethylamide (LiEDA) aggregates that were in good agreement with published experimental data.

Hydrodynamic Analysis of the Human Progesterone Receptor A-Isoform Reveals that Self-Association Occurs in the Micromolar Range

Human progesterone receptors exist as two functionally distinct isoforms, an 83 kDa A-receptor (PR-A) and a 99 kDa B-receptor (PR-B). The isoforms are identical except that PR-B has an additional 164 amino acids at its N-terminus. We have previously characterized the hydrodynamics and solution assembly energetics of PR-B [Heneghan, A. F., et al. (2005) Biochemistry 44, 9528-9537], and here we present an analysis of PR-A. At micromolar concentrations of the receptor, sedimentation velocity studies demonstrate that PR-A undergoes a concentration-dependent change in its sedimentation coefficient distribution, indicative of a self-associating system. Global analysis of data sets collected at multiple PR-A concentrations supports the presence of a hydrodynamically homogeneous 3.50 S monomer species in equilibrium with a 7.15 S dimer species. Sedimentation equilibrium analysis demonstrates that self-association can be rigorously described by a monomer-dimer assembly reaction and a dimerization free energy of -7.6 +/- 0.6 kcal/mol. Both the PR-A monomer and dimer are structurally asymmetric, although the extent of asymmetry is significantly decreased for the dimer, indicative of quaternary-induced hydrodynamic compaction. Limited proteolysis studies suggest that PR-A asymmetry arises from an ensemble of partially folded conformations within the N-terminal half of the molecule. Finally, comparison to our previous work on PR-B self-association energetics demonstrates that it dimerizes, under identical solution conditions, with an affinity at least 8-fold weaker than that of PR-A. Thus, residues unique to the B-isoform destabilize receptor assembly energetics. Importantly, the physical and chemical driving forces underlying isoform-specific dimerization suggest that B-unique amino acids modulate the energetics through an allosteric mechanism.

Structure of the Boronic Acid Dimer and the Relative Stabilities of Its Conformers

Despite the widespread use of boronic acids in materials science and as pharmaceutical agents, many aspects of their structure and reactivity are not well understood. In this research the boronic acid dimer, [HB(OH)(2)](2), was studied by second-order Møller-Plesset (MP2) perturbation theory and coupled cluster methodology with single and double excitations (CCSD). Pople split-valence 6-31+G*, 6-311G**, and 6-311++G** and Dunning-Woon correlation-consistent cc-pVDZ, aug-cc-pVDZ, cc-pVTZ, and aug-cc-pVTZ basis sets were employed for the calculations. A doubly hydrogen-bonded conformer (1) of the dimer was consistently found to be lowest in energy; the structure of 1 was planar (C(2h)) at most computational levels employed but was significantly nonplanar (C(2)) at the MP2/6-311++G** and CCSD/6-311++G** levels, the result of an intrinsic problem with Pople-type sp-diffuse basis functions on heavy atoms. The dimerization energy, enthalpy, and free energy for the formation of (1) from the exo-endo conformer of the monomer were -10.8, -9.2, and +1.2 kcal/mol, respectively, at the MP2/aug-cc-pVTZ level. Several other hydrogen-bonded conformers of the dimer were local minima on the potential energy surface (PES) and ranged from 2 to 5 kcal/mol higher in energy than 1. Nine doubly OH-bridged conformers, in which the boron atoms were tetracoordinated, were also local minima on the PES, but they were all greater than 13 kcal/mol higher in energy than 1; doubly H-bridged structures proved to be transition states. MP2 and CCSD results were compared to those from the BLYP, B3LYP, OLYP, O3LYP, PBE1PBE, and TPSS functionals with the 6-311++G** and aug-cc-pVTZ basis sets; the PBE1PBE functional performed best relative to the MP2 and CCSD results. Self-consistent reaction field (SCRF) calculations predict that boronic acid dimerization is less favorable in solution than in vacuo.

Self-Association Energetics of an Intact, Full-Length Nuclear Receptor: The B-Isoform of Human Progesterone Receptor Dimerizes in the Micromolar Range

We are focused on understanding the mechanisms underlying eukaryotic gene regulation, using the human progesterone receptor (PR) and its interactions with its DNA response elements as a model system. An understanding of PR function is complicated by the presence of two transcriptionally distinct isoforms, an 83 kDa A-receptor (PR-A) and a 99 kDa B-receptor (PR-B). The two isoforms are identical except the B-receptor contains an additional 164 residues at its N-terminus. As a first step toward understanding the principles by which the two isoforms assemble at complex promoters, we examined the energetics of PR-B self-association using sedimentation velocity and sedimentation equilibrium methods. Full-length human PR-B was purified to 95% homogeneity from baculovirus-infected insect cells. Using a fluorescence hormone binding assay, we determined the purified protein to be highly active in its ability to bind ligand. Sedimentation velocity studies of hormone-bound PR-B at pH 8.0, 4 degrees C, and 50 mM NaCl demonstrate that it undergoes a concentration-dependent change in its sedimentation coefficient, existing as a 4.0S species at submicromolar concentrations but forming a 5.7S species at higher concentrations. These results strongly suggest that PR-B undergoes self-association in the micromolar range. This hypothesis was examined rigorously using sedimentation equilibrium. Global analysis of the sedimentation equilibrium data demonstrated that PR-B self-association was well described by a monomer-dimer model with a dimerization free energy of -7.2 +/- 0.7 kcal/mol. The role of NaCl in regulating PR-B dimerization was examined by carrying out sedimentation velocity and equilibrium studies under high salt conditions. At 300 mM NaCl, PR-B is exclusively monomeric in the micromolar range, thus revealing a significant ionic contribution to the assembly energetics. Further, the monomer sediments as a structurally homogeneous, but highly asymmetric, 4.0S species. Limited proteolysis of PR-B demonstrated that the hydrodynamic asymmetry is due in part to an extended, nonglobular conformation localized to the N-terminal region of PR-B. In contrast, the DNA binding domain (DBD) and hormone binding domain (HBD) exist as independent structural units, and the activation function N-terminal to the DBD (AF-1) shows moderate structure. These results represent the first rigorous analysis of the self-assembly energetics of an intact nuclear receptor and suggest that PR function is more complex than envisioned by traditional models.

Insights into the Recognition and Association of Transmembrane α-Helices. The Free Energy of α-Helix Dimerization in Glycophorin A

The free energy of alpha-helix dimerization of the transmembrane (TM) region of glycophorin A was estimated from a 125-ns molecular dynamics (MD) simulation in a membrane mimetic. The free energy profile was obtained by allowing the TM helical segments to diffuse reversibly along the reaction pathway. Partition of the potential of mean force into free energy components illuminates the critical steps of alpha-helix recognition and association. At large separations, the TM segments are pushed together by the solvent, allowing initial, but not necessarily native, interhelical interactions to occur. This early recognition stage precedes the formation of native contacts, which is accompanied by a tilt of the helices, characteristic of the dimeric structure. This step is primarily driven by the van der Waals helix-helix interactions. Free energy perturbation calculations of the L75A and I76A point mutations reveal a disruption in helix-helix association due to a loss of favorable dispersion interactions. Additional MD simulations of the native TM dimer and of a single alpha-helix confirm that, prior to association, individual alpha-helices are independently stable, in agreement with the "two-stage" model of integral membrane protein folding.