Dimerization Free Energy Research Articles

Construction, Validation, and Application of Digital Oil: Investigation of Asphaltene Association Toward Asphaltene-Precipitation Prediction

Summary Digital oil, a realistic molecular model of crude oil for a target reservoir, opens a new door to understand properties of crude oil under a wide range of thermodynamic conditions. In this study, we constructed a digital oil to model a light crude oil using analytical experiments after separating the light crude oil into gas, light and heavy fractions, and asphaltenes. The gas and light fractions were analyzed by gas chromatography (GC), and 105 kinds of molecules, including normal alkanes, isoalkanes, naphthenes, alkylbenzenes, and polyaromatics (with a maximum of three aromatic rings), were directly identified. The heavy fraction and asphaltenes were analyzed by elemental analysis, molecular-weight (MW) measurement with gel-permeation chromatography (GPC), and hydrogen and carbon nuclear-magnetic-resonance (NMR) spectroscopy, and represented by the quantitative molecular-representation method, which provides a mixture model imitating distributions of the crude-oil sample. Because of the low weight concentration of asphaltenes in the light crude oil (approximately 0.1 wt%), the digital oil model was constructed by mixing the gas, light-, and heavy-fraction models. To confirm the validity of the digital oil, density and viscosity were calculated over a wide range of pressures at the reservoir temperature by molecular-dynamics (MD) simulations. Because only experimental data for the liquid phase were available, we predicted the liquid components of the digital oil at pressures lower than 16.3 MPa (i.e., the bubblepoint pressure) by flash calculation, and calculated the liquid density by MD simulation. The calculated densities coincided with the experimental values at all pressures in the range from 0.1 to 29.5 MPa. We calculated the viscosity of the liquid phase at the same pressures by two independent methods. The calculated viscosities were in good agreement with each other. Moreover, the viscosity change with pressure was consistent with the experimental data. As a step for application of digital oil to predict asphaltene-precipitation risk, we calculated dimerization free energy of asphaltenes (which we regarded as asphaltene self-association energy) in the digital oil at the reservoir condition, using MD simulation with the umbrella sampling method. The calculated value is consistent with reported values used in phase-equilibrium calculation. Digital oil is a powerful tool to help us understand mechanisms of molecular-scale phenomena in oil reservoirs and solve problems in the upstream and downstream petroleum industry.

Free energy simulations for protein ligand binding and stability

We summarize several computational techniques to determine relative free energies for condensed-phase systems. The focus is on practical considerations which are capable of making direct contact with experiments. Particular applications include the thermodynamic stability of apo- and holo-myoglobin, insulin dimerization free energy, ligand binding in lysozyme, and ligand diffusion in globular proteins. In addition to provide differential free energies between neighboring states, converged umbrella sampling simulations provide insight into migration barriers and ligand dissociation barriers and analysis of the trajectories yield additional insight into the structural dynamics of fundamental processes. Also, such simulations are useful tools to quantify relative stability changes for situations where experiments are difficult. This is illustrated for NO-bound myoglobin. For the dissociation of benzonitrile from lysozyme it is found that long umbrella sampling simulations are required to approximately converge the free energy profile. Then, however, the resulting differential free energy between the bound and unbound state is in good agreement with estimates from molecular mechanics with generalized Born surface area simulations. Furthermore, comparing the barrier height for ligand escape suggests that ligand dissociation contains a non-equilibrium component.

A model-free method for measuring dimerization free energies of CLC-ec1 in lipid bilayers.

The thermodynamic reasons why membrane proteins form stable complexes inside the hydrophobic lipid bilayer remain poorly understood. This is largely because of a lack of membrane-protein systems amenable for equilibrium studies and a limited number of methods for measuring these reactions. Recently, we reported the equilibrium dimerization of the CLC-ec1 Cl-/H+ transporter in lipid bilayers (Chadda et al. 2016. eLife https://doi.org/10.7554/eLife.17438), which provided a new type of model system for studying protein association in membranes. The measurement was conducted using the subunit-capture approach, involving passive dilution of the protein in large multilamellar vesicles, followed by single-molecule photobleaching analysis of the Poisson distribution describing protein encapsulation into extruded liposomes. To estimate the fraction of dimers (FDimer ) as a function of protein density, the photobleaching distributions for the nonreactive, ideal monomer and dimer species must be known so that random co-capture probabilities can be accounted for. Previously, this was done by simulating the Poisson process of protein reconstitution into a known size distribution of liposomes composed of Escherichia coli polar lipids (EPLs). In the present study, we investigate the dependency of FDimer and ΔG° on the modeling through a comparison of different liposome size distributions (EPL versus 2:1 POPE/POPG). The results show that the estimated FDimer values are comparable, except at higher densities when liposomes become saturated with protein. We then develop empirical controls to directly measure the photobleaching distributions of the nonreactive monomer (CLC-ec1 I201W/I422W) and ideal dimer (WT CLC-ec1 cross-linked by glutaraldehyde or CLC-ec1 R230C/L249C cross-linked by a disulfide bond). The measured equilibrium constants do not depend on the correction method used, indicating the robustness of the subunit-capture approach. This strategy therefore presents a model-free way to quantify protein dimerization in lipid bilayers, offering a simplified strategy in the ongoing effort to characterize equilibrium membrane-protein reactions in membranes.

NMR and DFT Studies of 2-Oxo-1,2,3,4-tetrahydropyridines: Solvent and Temperature Effects

Various 5-carboethoxy-2-oxo-1,2,3,4-tetrahydropyridines and their corresponding oxidation products containing methoxy or nitro groups on different positions of the C4-aryl ring were synthesized and the effect of steric and electrostatic interactions of these aryl substituents on the characteristic peaks in 1H NMR spectra were investigated. In addition, the intermolecular interaction of the parent compound and its oxidized form with solvent was experimentally investigated. For this, 1H NMR spectra of these compounds at different concentrations and temperatures in [D6]DMSO and CDCl3 were investigated. For comparison of the dimerization ability of these heterocyclic compounds with different conformations, the binding electronic energies, the total enthalpies and free energies of dimerization in the gas and solution phases, and the QTAIM (quantum theory of atoms-in-molecules) analysis were determined. These interactions were also studied using density functional theory at the B3LYP/6–311++G(d,p) level. The theoretical results are in good agreement with the experimental results and indicate that the electronic effect of the methoxy and nitro groups on the C4-aryl ring influences the electron density of the heterocyclic ring via the σ bond and, consequently, the chemical shift of the heterocyclic ring protons.

Sequence-nonspecific Stabilization of Transmembrane Helical Peptide Dimer in Lipid Raft-like Bilayers in Atomistic Simulations. I. Dimerization Free Energy and Impact of Lipid-peptide Potential Energy

Sequence-nonspecific Stabilization of Transmembrane Helical Peptide Dimer in Lipid Raft-like Bilayers in Atomistic Simulations. I. Dimerization Free Energy and Impact of Lipid-peptide Potential Energy

Excessive aggregation of membrane proteins in the Martini model.

The coarse-grained Martini model is employed extensively to study membrane protein oligomerization. While this approach is exceptionally promising given its computational efficiency, it is alarming that a significant fraction of these studies demonstrate unrealistic protein clusters, whose formation is essentially an irreversible process. This suggests that the protein–protein interactions are exaggerated in the Martini model. If this held true, then it would limit the applicability of Martini to study multi-protein complexes, as the rapidly clustering proteins would not be able to properly sample the correct dimerization conformations. In this work we first demonstrate the excessive protein aggregation by comparing the dimerization free energies of helical transmembrane peptides obtained with the Martini model to those determined from FRET experiments. Second, we show that the predictions provided by the Martini model for the structures of transmembrane domain dimers are in poor agreement with the corresponding structures resolved using NMR. Next, we demonstrate that the first issue can be overcome by slightly scaling down the Martini protein–protein interactions in a manner, which does not interfere with the other Martini interaction parameters. By preventing excessive, irreversible, and non-selective aggregation of membrane proteins, this approach renders the consideration of lateral dynamics and protein–lipid interactions in crowded membranes by the Martini model more realistic. However, this adjusted model does not lead to an improvement in the predicted dimer structures. This implicates that the poor agreement between the Martini model and NMR structures cannot be cured by simply uniformly reducing the interactions between all protein beads. Instead, a careful amino-acid specific adjustment of the protein–protein interactions is likely required.

Can Simple Interaction Models Explain Sequence-Dependent Effects in Peptide Homodimerization?

The development of rapid methods to explain and predict peptide interactions, aggregation, and self-assembly has become important to understanding amyloid disease pathology, the shelf stability of peptide therapeutics, and the design of novel peptide materials. Although experimental aggregation databases have been used to develop correlative and statistical models, molecular simulations offer atomic-level details that potentially provide greater physical insight and allow one to single out the most explanatory simple models. Here, we outline one such approach using a case study that develops homodimerization models for serine-glycine peptides with various hydrophobic leucine mutations. Using detailed all-atom simulations, we calculate reference dimerization free energy profiles and binding constants for a small peptide library. We then use statistical methods to systematically assess whether simple interaction models, which do not require expensive simulations and free energy calculation, can capture them. Surprisingly, some combinations of a few simple scaling laws well recapitulate the detailed, all-atom results with high accuracy. Specifically, we find that a recently proposed phenomenological hydrophobic force law and coarse measures of entropic effects in binding offer particularly high explanatory power, underscoring the physical relevance to association that these driving forces can play.

The Significance of Surface Complementarity on the Free Energy of Membrane Protein Assembly in Membranes

The dimerization interface of the 18-TM helix ClC-ec1 Cl-/H+ antiporter is formed by four alpha helices (H, I, P and Q) that are lined entirely by non-polar residues (leucines and isoleucines). The resultant surface fits well with its protein partner, presenting ClC-ec1 as an interesting model system to study the effect of shape complementarity on the stability of protein association. Recently, we established single-molecule fluorescence microscopy methods allowing for the measurement of the equilibrium dimerization reaction of ClC-ec1 in 2:1 POPE/POPG lipid bilayers. Here, we investigate the effect that cavities, engineered via subtractive substitutions to ALA, have on the free energy of dimerization in the lipid bilayer. We first engineered ClC constructs with large cavities by mutating entire helix surfaces to ALA, 4-5 residues at a time. Each of these constructs were stable and folded upon purification in detergent, and demonstrated comparable functional Cl- transport after reconstitution in membranes. However, while all-ALA helix I, P and Q are almost entirely dimeric as determined by size exclusion chromatography in detergent, all-ALA helix H shifts to a monomeric form. Further investigation of the residues on helix H indicates that L194A alone is responsible for the shift to the monomeric state. Cysteine accessibility and functional reconstitution experiments show that L194A is folded and functional in both monomeric and dimeric states. Measurement of the ΔΔG of dimerization in 2:1 POPE/POPG shows that L194A shifts stability by 2 kcal/mole, similar to the effect of a bulky I422W substitution at the interface. We conclude that L194 is an energetic hotspot on the dimerization interface, but the reason for this shift in stability remains unknown. Examination of the structure shows that L194 is positioned along the symmetry interface of the subunit fold suggesting that it may be involved in defining the shape of the dimerization interface, and thus mutation may alter shape complementarity by increasing dynamics of those helices.

Measuring the Free Energy of ClC-ec1 Dimerization in Membranes using Single Molecule Photobleaching Analysis

Why do greasy membrane proteins interact with their greasy protein partners instead of the similarly greasy lipid bilayer? This is a question at the root of membrane protein oligomerization and folding in membranes. Recently, we turned to the homodimeric ClC-ec1 Cl-/H+ antiporter, in order to develop a new model system that can be used to investigate this question. The ClC-ec1 dimerization interface is large (1200 A2) and lined by ∼20 non-polar residues, forming an interaction surface exhibiting high shape complementarity. Previous studies showed that ClC-ec1 is folded and functional in both monomeric and dimeric states. Using a single-molecule microscopy approach, we measured the photobleaching distributions across a wide range of subunit/lipid dilutions, with the lowest representing a sub-biological condition of 1 subunit per 40 E. coli membranes. The data shows that wild-type ClC-ec1 follows a monomer to dimer reaction that is reversible and fits to an equilibrium isotherm, allowing for determination of the dimerization free energy in 2:1 POPE/POPG lipid bilayers (Chadda et al., eLife 2016). Next, two approaches were used to investigate how residues at the interaction interface influence the free energy of dimerization. First, bulky TRP residues were introduced resulting in a destabilization of the dimer by 2 kcal/mole per TRP. Second, subtractive substitutions to ALA were constructed introducing cavities at the interface. While most of the ALA substitutions appear to have no effect, L194A destabilizes the dimer by 2 kcal/mole in lipid bilayers while maintaining both fold and function. These studies present ClC-ec1 as a robust model system for probing the physical forces driving protein association in membranes.

Dimerization and conformation-related free energy landscapes of dye-tagged amyloid-β12-28 linked to FRET experiments.

We have investigated the free energy landscape of Aβ-peptide dimer models in connection to gas-phase FRET experiments. We use a FRET-related distance coordinate and one conformation-related coordinate per monomer for accelerated structural exploration with well-tempered metadynamics in solvent and in vacuo. The free energy profiles indicate that FRET under equilibrium conditions should be significantly affected by the de-solvation upon the transfer of ions to the gas-phase. In contrast, a change in the protonation state is found to be less impacting once de-solvated. Comparing F19P and WT alloforms, for which we measure different FRET efficiencies in the gas-phase, we predict only the relevant structural differences in the solution populations, not under gas-phase equilibrium conditions. This finding supports the hypothesis that the gas-phase action-FRET measurement after ESI operates under non-equilibrium conditions, with a memory of the solution conditions - even for the dimer of this relatively short peptide. The structural differences in solution are rationalized in terms of conformational propensities around residue 19, which show a transition to a poly-proline type of pattern upon mutation to F19P - a difference that gets lost in the gas-phase.

Convergence and Sampling in Determining Free Energy Landscapes for Membrane Protein Association.

Potential of mean force (PMF) calculations are used to characterize the free energy landscape of protein–lipid and protein–protein association within membranes. Coarse-grained simulations allow binding free energies to be determined with reasonable statistical error. This accuracy relies on defining a good collective variable to describe the binding and unbinding transitions, and upon criteria for assessing the convergence of the simulation toward representative equilibrium sampling. As examples, we calculate protein–lipid binding PMFs for ANT/cardiolipin and Kir2.2/PIP2, using umbrella sampling on a distance coordinate. These highlight the importance of replica exchange between windows for convergence. The use of two independent sets of simulations, initiated from bound and unbound states, provide strong evidence for simulation convergence. For a model protein–protein interaction within a membrane, center-of-mass distance is shown to be a poor collective variable for describing transmembrane helix–helix dimerization. Instead, we employ an alternative intermolecular distance matrix RMS (DRMS) coordinate to obtain converged PMFs for the association of the glycophorin transmembrane domain. While the coarse-grained force field gives a reasonable Kd for dimerization, the majority of the bound population is revealed to be in a near-native conformation. Thus, the combination of a refined reaction coordinate with improved sampling reveals previously unnoticed complexities of the dimerization free energy landscape. We propose the use of replica-exchange umbrella sampling starting from different initial conditions as a robust approach for calculation of the binding energies in membrane simulations.

Dissipative Particle Dynamics Study on the Aggregation Behavior of Asphaltenes under Shear Fields

In the present work, the effects of shear fields on the aggregation of asphaltene molecules in heptane were investigated by means of dissipative particle dynamics simulations. The geometries of asphaltene aggregates without shear fields were studied, and the simulation results provide an interpretation of the experimental results on the microscopic level. The effects of shear fields on asphaltene aggregates were also investigated by accessing the radial distribution functions, spatial orientation correlation functions, and the radii of gyrations. We show that the shear fields can destroy the conformational order of the aggregates by damaging the organized structure and isolating the asphaltenes. As the radius of gyration results show, the asphaltene molecules are elongated to be alike-polymers by shear fields. Moreover, the reason why the viscosity decreases under shear fields is that the shear fields lead to the increase of dimerization free energies.

A Comprehensive Description of the Homo and Heterodimerization Mechanism of the Chemokine Receptors CCR5 and CXCR4

Signal transduction across cellular membranes is controlled by G protein-coupled receptors (GPCRs). In this family of transmembrane proteins the binding of extracellular physiological ligands stabilise the active conformation of receptors, leading to cellular response. It is widely accepted that members of the largest GPCR family self-assemble as dimers or higher-order but the functional consequences of the dimerization was described only for few receptors. The chemokines receptors are GPCRs implicated in many physiological processes, including the functioning and maintenance of the immune system. These receptors represent prime targets for therapeutic intervention in a wide spectrum of inflammatory and autoimmune diseases, heart diseases, and HIV. The CXCR4 and CCR5 receptors are two of the manly studied playing crucial roles in different pathologies. It was recently shown that inhibition of the CCR5-CXCR4 heterodimer formation reduces atherosclerosis in a hyperlipidemic mouse model. Furthermore the entry of HIV-1 virus into host cells requires CXCR4 and CCR5 in addition to its main receptor CD4. In this scenario the use of computational techniques able to describe complex biological processes such as protein dimerization acquires a great importance. Combining coarse-grained (CG) molecular dynamics and well-tempered metadynamics (MetaD) we are able to describe the mechanism of dimer formation, capturing multiple association and dissociation events allowing to compute a detailed free energy landscape of the process. CG-MetaD is an enhanced sampling method particularly suitable to describe processes with very slow rates of interconversion among the possible states of the system (i.e. protein dimerization). This approach provides an accurate and comprehensive description of the dimerization free energy landscape, thereby revealing critical motions and important structural-dynamical features (i.e. transient complex, role of lipids) involved in the homo and heterodimer formation of the CCR5 and CXCR4 receptors.

Heat Capacity Changes and Disorder-to-Order Transitions in Allosteric Activation.

Allosteric coupling in proteins is ubiquitous but incompletely understood, particularly in systems characterized by coupling over large distances. Binding of the allosteric effector, bio-5'-AMP, to the Escherichia coli biotin protein ligase, BirA, enhances the protein's dimerization free energy by -4 kcal/mol. Previous studies revealed that disorder-to-order transitions at the effector binding and dimerization sites, which are separated by 33 Å, are integral to functional coupling. Perturbations to the transition at the ligand binding site alter both ligand binding and coupled dimerization. Alanine substitutions in four loops on the dimerization surface yield a range of energetic effects on dimerization. A glycine to alanine substitution at position 142 in one of these loops results in a complete loss of allosteric coupling, disruption of the disorder-to-order transitions at both functional sites, and a decreased affinity for the effector. In this work, allosteric communication between the effector binding and dimerization surfaces in BirA was further investigated by performing isothermal titration calorimetry measurements on nine proteins with alanine substitutions in three dimerization surface loops. In contrast to BirAG142A, at 20 °C all variants bind to bio-5'-AMP with free energies indistinguishable from that measured for wild-type BirA. However, the majority of the variants exhibit altered heat capacity changes for effector binding. Moreover, the ΔCp values correlate with the dimerization free energies of the effector-bound proteins. These thermodynamic results, combined with structural information, indicate that allosteric activation of the BirA monomer involves formation of a network of intramolecular interactions on the dimerization surface in response to bio-5'-AMP binding at the distant effector binding site.

Predicting RNA Duplex Dimerization Free-Energy Changes upon Mutations Using Molecular Dynamics Simulations.

The dimerization free energies of RNA-RNA duplexes are fundamental values that represent the structural stability of RNA complexes. We report a comparative analysis of RNA-RNA duplex dimerization free-energy changes upon mutations, estimated from a molecular dynamics simulation and experiments. A linear regression for nine pairs of double-stranded RNA sequences, six base pairs each, yielded a mean absolute deviation of 0.55 kcal/mol and an R(2) value of 0.97, indicating quantitative agreement between simulations and experimental data. The observed accuracy indicates that the molecular dynamics simulation with the current molecular force field is capable of estimating the thermodynamic properties of RNA molecules.

Generalized Born and Explicit Solvent Models for Free Energy Calculations in Organic Solvents: Cyclodextrin Dimerization.

Evaluation of solvation (binding) free energies with implicit solvent models in different dielectric environments for biological simulations as well as high throughput ligand screening remain challenging endeavors. In order to address how well implicit solvent models approximate explicit ones we examined four generalized Born models (GB(Still), GB(HCT), GB(OBC)I, and GB(OBC)II) for determining the dimerization free energy (ΔG(0)) of β-cyclodextrin monomers in 17 implicit solvents with dielectric constants (D) ranging from 5 to 80 and compared the results to previous free energy calculations with explicit solvents ( Zhang et al. J. Phys. Chem. B 2012 , 116 , 12684 - 12693 ). The comparison indicates that neglecting the environmental dependence of Born radii appears acceptable for such calculations involving cyclodextrin and that the GB(Still) and GB(OBC)I models yield a reasonable estimation of ΔG(0), although the details of binding are quite different from explicit solvents. Large discrepancies between implicit and explicit solvent models occur in high-dielectric media with strong hydrogen bond (HB) interruption properties. ΔG(0) with the GB models is shown to correlate strongly to 2(D-1)/(2D+1) (R(2) ∼ 0.90) in line with the Onsager reaction field ( Onsager J. Am. Chem. Soc. 1936 , 58 , 1486 - 1493 ) but to be very sensitive to D (D < 10) as well. Both high-dielectric environments where hydrogen bonds are of interest and low-dielectric media such as protein binding pockets and membrane interiors therefore need to be considered with caution in GB-based calculations. Finally, a literature analysis of Gibbs energy of solvation of small molecules in organic liquids shows that the Onsager relation does not hold for real molecules since the correlation between ΔG(0) and 2(D-1)/(2D+1) is low for most solutes. Interestingly, explicit solvent calculations of the solvation free energy ( Zhang et al. J. Chem. Inf. Model . 2015 , 55 , 1192 - 1201 ) reproduce the weak experimental correlations with 2(D-1)/(2D+1) very well.

Adaptable Lipid Matrix Promotes Protein-Protein Association in Membranes.

The cell membrane is "stuffed" with proteins, whose transmembrane (TM) helical domains spontaneously associate to form functionally active complexes. For a number of membrane receptors, a modulation of TM domains' oligomerization has been shown to contribute to the development of severe pathological states, thus calling for detailed studies of the atomistic aspects of the process. Despite considerable progress achieved so far, several crucial questions still remain: How do the helices recognize each other in the membrane? What is the driving force of their association? Here, we assess the dimerization free energy of TM helices along with a careful consideration of the interplay between the structure and dynamics of protein and lipids using atomistic molecular dynamics simulations in the hydrated lipid bilayer for three different model systems - TM fragments of glycophorin A, polyalanine and polyleucine peptides. We observe that the membrane driven association of TM helices exhibits a prominent entropic character, which depends on the peptide sequence. Thus, a single TM peptide of a given composition induces strong and characteristic perturbations in the hydrophobic core of the bilayer, which may facilitate the initial "communication" between TM helices even at the distances of 20-30 Å. Upon tight helix-helix association, the immobilized lipids accommodate near the peripheral surfaces of the dimer, thus disturbing the packing of the surrounding. The dimerization free energy of the modeled peptides corresponds to the strength of their interactions with lipids inside the membrane being the lowest for glycophorin A and similarly higher for both homopolymers. We propose that the ability to accommodate lipid tails determines the dimerization strength of TM peptides and that the lipid matrix directly governs their association.

Supramolecular polymerization: a coarse grained molecular dynamics study.

A coarse-grained (CG) force field to model the self-assembly of benzene-1,3,5-tricarboxamide (BTA) class of compounds in nonpolar solvents has been developed. The model includes an intrinsic point dipole embedded on one of the CG beads so as to impart a macrodipole moment to the oligomer, one of its characteristic feature. Chemical specificity has been preserved by benchmarking against results, including dimerization and solvation free energies, obtained from an all-atom representation. Starting from a well-dispersed configuration in n-nonane, BTA molecules self-assemble to form one-dimensional stacks. Free energy (FE) changes for the various manner in which short oligomers can exchange between the assembled and the dispersed states have been calculated. These calculations show BTA to self-assemble via a downhill cooperative mechanism with a nucleus size of three.

Critical Role of a Glycine Residue in an Allosteric Switch

Thermodynamic coupling in proteins is ubiquitous but mechanistically poorly understood, particularly for systems in which the coupling occurs over large distances. In the E. coli biotin repressor the coupling between homodimerization and allosteric effector, bio-5′-AMP, is −4.0 kcal/mol. The two coupled processes occur at sites separated by 33A. Structural and thermodynamic studies indicate that the coupling is accompanied by disorder-to-order transitions at the two distant functional sites. Perturbations to the transition at the ligand binding site via alanine substitutions alter both ligand binding and coupled dimerization. Alanine substitutions in four loops in the dimerization surface yield a range of energetic consequences for BirA dimerization. For one of these variants, BirAG142A, the free energies of dimerization and corepressor binding are consistent with complete abolition of coupling. Structural studies of the variant indicate that the loss of coupling is accompanied by disruption of the disorder-to-order transitions at both functional surfaces. In this work the structural basis of coupling between the dimerization and ligand binding surfaces was further investigated by measuring the consequences of alanine substitutions distributed in three of the dimerization loops on corepressor binding. Isothermal titration calorimetry measurements indicate that, in contrast to BirAG142A, the variants, several of which dimerize very weakly, all bind to bio-5′-AMP with energetics indistinguishable from wild-type BirA. The results indicate that a single glycine residue serves as a switch for allosteric regulation of BirA.

Determining the Free Energy of Membrane Protein Dimerization in Lipid Bilayers

Experimental determination of the free energy of membrane protein oligomerization in lipid bilayers is difficult. There are few membrane protein systems that allow for this equilibrium measurement, and for strong complexes, the amount of dilution necessary to observe dissociation often obscures experimental detection. We have devised a new model system for measuring equilibrium dimerization in membranes, using the homodimeric CLC-ec1 Cl-/H+ antiporter. A mutated version of CLC-ec1 that bears a single tryptophan substitution on the dimerization interface (CLC-W) shifts the protein to the monomeric state in detergent micelles. We quantitatively labelled CLC-W with Cy3 or Cy5 fluorophore and reconstituted the protein into 2:1 POPE/POPG lipids. Then, we measured macroscopic Forster resonance energy transfer (FRET) in large membranes to assess CLC-W dimerization. Mixing CLC-W-Cy3 and CLC-W-Cy5 in detergent before reconstitution, or fusing CLC-W-Cy3 liposomes with CLC-W-Cy5 liposomes yields a FRET signal indicative of dimer formation and equilibrium exchange. We also confirmed that the protein was folded, by measuring Cl- transport function. We then investigated dissociation of CLC-W by “traditional” dilution in membranes. Since wild-type CLC-ec1 does not undergo dimer exchange, we used CLC-ec1-Cy3 mixed with CLC-ec1-Cy5 to determine the “all-monomer” background signal, and co-labelled CLC-ec1-Cy3/Cy5 to determine the “all-dimer” FRET signal, at various Cy3:Cy5 labeling ratios and protein:lipid densities. With this, we observe that CLC-W begins to dissociate at a density of 1 protein per 300,000 lipids. At densities below 1 protein per 650,000 lipids, macroscopic FRET is obscured by background scattering and so we turn to single-molecule fluorescence microscopy to measure CLC-W stoichiometry by fluorophore photo-bleaching. These studies demonstrate that we have pushed the observable limit of this reaction, expanding our ability to measure the free energy of membrane protein assembly in lipid bilayers.