Dimer Equilibrium Research Articles

MP4 Study of the Anharmonic Coupling of the Shared Proton Stretching Vibration of the Protonated Water Dimer in Equilibrium and Transition States.

The structure and harmonic and anharmonic IR spectra of the protonated water dimer (PWD) were calculated in C1, C2, and Cs symmetry at the MP4/acc-pVTZ level of theory. We found that structure and IR spectra are practically identical in C2 and C1 symmetry, demonstrating that an equilibrium C1 configuration of the PWD is not realized. Anharmonic coupling of the shared proton stretching vibration with all other modes in the PWD in C2 and Cs symmetry was the focus of this investigation. For this purpose, 28 two-dimensional potential energy surfaces (2D PES) were built at the MP4/acc-pVTZ level of theory and the corresponding vibrational Schrödinger equations were solved using the DVR method. Differences in the coupling of the investigated mode with other modes in the C2 and Cs configurations, along with some factors that determine the red- or blue-shift of the stretching vibration frequency, were analyzed. We obtained a rather reasonable value of the stretching frequency of the bridging proton (1058.4 cm-1) unperturbed by Fermi resonance. The Fermi resonance between the fundamental vibration ν7 and the combined vibration ν2 + ν6 of the same symmetry was analyzed through anharmonic second-order perturbation theory calculations, as well as by 3D PES constructed using Q2, Q6, and Q7 as normal coordinates. A significant (up to 50%) transfer of intensity from the fundamental vibration to the combined one was found. We have estimated the frequency of the bridging proton stretching vibration in the Cs configuration of the PWD based on calculations of the intrinsic anharmonicity and anharmonic double modes interactions at the MP4/acc-pVTZ level of theory (1261 cm-1).

Membranes Matter: Predicting Drug Toxicity

It remains a challenge to predict whether a new drug candidate will have undesirable side effects. Many biologically active molecules, including drugs and drug-leads, are amphiphiles that partition into lipid bilayers, which will tend to alter bilayer physical properties, thereby modulating membrane protein function. Such bilayer-modifying molecules may be promiscuous modifiers of membrane protein function, raising the possibility that they have off-target effects. Consequently, it may be possible to predict whether a compound will have off-target effects based on quantitative studies on the compound's bilayer-modifying potential. To address this question, we developed an assay to quantify the bilayer-modifying potential of large numbers of compounds using a gramicidin-based fluorescence assay (GBFA), which reports how a compound alters the gramicidin monomer ↔ dimer equilibrium. Using this assay, we have shown that many drug and drug-leads alter lipid bilayer properties at the concentrations where these compounds become indiscriminate modifiers of membrane protein function. Such indiscriminate modifiers of membrane protein function are likely to have off target effects; we pursued this question in a study on libraries of compounds that had been tested for toxicity in “high-content” screening assays that quantified cellular ATP levels, nuclear morphology, nuclear membrane integrity, and apoptosis in immortalized liver, lung, and neuronal cell lines. We tested 524 total compounds comprising four libraries (one non-toxic library and three mixed libraries of non-toxic and toxic compounds; the libraries were “blinded” until the results of the GBFA were known) and found that the GBFA predicts cellular toxicity, with minimal assignment of false positives. These results suggest that in vitro measurement could be used as a warning sign for off-target biological effects in drug discovery efforts and, further, provides a mechanism by which amphiphiles exert their toxicity, namely by altering lipid bilayer physical properties.

Hemoglobin Kirklareli (α H58L), a New Variant Associated with Iron Deficiency and Increased CO Binding

Mutations in hemoglobin can cause a wide range of phenotypic outcomes, including anemia due to protein instability and red cell lysis. Uncovering the biochemical basis for these phenotypes can provide new insights into hemoglobin structure and function as well as identify new therapeutic opportunities. We report here a new hemoglobin α chain variant in a female patient with mild anemia, whose father also carries the trait and is from the Turkish city of Kirklareli. Both the patient and her father had a His-58(E7) → Leu mutation in α1. Surprisingly, the patient's father is not anemic, but he is a smoker with high levels of HbCO (∼16%). To understand these phenotypes, we examined recombinant human Hb (rHb) Kirklareli containing the α H58L replacement. Mutant α subunits containing Leu-58(E7) autoxidize ∼8 times and lose hemin ∼200 times more rapidly than native α subunits, causing the oxygenated form of rHb Kirklareli to denature very rapidly under physiological conditions. The crystal structure of rHb Kirklareli shows that the α H58L replacement creates a completely apolar active site, which prevents electrostatic stabilization of bound O2, promotes autoxidation, and enhances hemin dissociation by inhibiting water coordination to the Fe(III) atom. At the same time, the mutant α subunit has an ∼80,000-fold higher affinity for CO than O2, causing it to rapidly take up and retain carbon monoxide, which prevents denaturation both in vitro and in vivo and explains the phenotypic differences between the father, who is a smoker, and his daughter.

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.

Desolvation and aggregation of sterically demanding alkali metal diarylphosphides.

The reaction between (Dipp)2PH and one equivalent of n-BuLi, PhCH2Na or PhCH2K in THF gives the complexes [(Dipp)2P]Li(THF)3 (2a), {[(Dipp)2P]Na(THF)2}2 (3a) and [(Dipp)2P]K(THF)4 (4a), respectively [Dipp = 2,6-iPr2C6H3]. Exposure of these compounds to vacuum yields the alternative solvates [(Dipp)2P]Li(THF)2 (2b), [(Dipp)2P]Na(THF)1.5 (3b), and [(Dipp)2P]K (4b), respectively; the alternative adduct [(Dipp)2P]Na(PMDETA) (3c) was prepared by treatment of 3a with PMDETA. Treatment of (Dipp)(Mes)PH or (Mes)2PH with one equivalent of n-BuLi in THF gives the complexes [(Dipp)(Mes)P]Li(THF)3 (7a) and [(Mes)2P]2Li2(THF)2(OEt2) (8a) after crystallisation from diethyl ether [Mes = 2,4,6-Me3C6H2]; crystallisation of 8a from hexane gives the alternative adduct [(Mes)2P]Li(THF)3 (8b). Exposure of 7a, 8a and 8b to vacuum leads to loss of coordinated solvent, yielding the solvates [(Dipp)(Mes)P]Li(THF)2 (7b) and [(Mes)2P]Li(THF) (8c). The solid-state structures of complexes 2a, 3a, 3c, 4a, 7a, 8a, and 8b have been determined by X-ray crystallography. Variable-temperature 31P{1H} and 7Li NMR spectroscopy indicates that 2b, 3b and 7b are subject to a monomer-dimer equilibrium in solution, where the monomeric forms are favoured at low temperature. In contrast, variable-temperature 31P{1H} and 7Li NMR spectroscopy suggests that 8c is subject to a dynamic equilibrium between a dimer and a cyclic trimer in solution, where the trimer is favoured at low temperatures.

Quaternary structures of opsin in live cells revealed by FRET spectrometry.

Rhodopsin is a prototypical G-protein-coupled receptor (GPCR) that initiates phototransduction in the retina. The receptor consists of the apoprotein opsin covalently linked to the inverse agonist 11-cis retinal. Rhodopsin and opsin have been shown to form oligomers within the outer segment disc membranes of rod photoreceptor cells. However, the physiological relevance of the observed oligomers has been questioned since observations were made on samples prepared from the retina at low temperatures. To investigate the oligomeric status of opsin in live cells at body temperatures, we utilized a novel approach called Förster resonance energy transfer spectrometry, which previously has allowed the determination of the stoichiometry and geometry (i.e. quaternary structure) of various GPCRs. In the current study, we have extended the method to additionally determine whether or not a mixture of oligomeric forms of opsin exists and in what proportion. The application of this improved method revealed that opsin expressed in live Chinese hamster ovary (CHO) cells at 37°C exists as oligomers of various sizes. At lower concentrations, opsin existed in an equilibrium of dimers and tetramers. The tetramers were in the shape of a near-rhombus. At higher concentrations of the receptor, higher-order oligomers began to form. Thus, a mixture of different oligomeric forms of opsin is present in the membrane of live CHO cells and oligomerization occurs in a concentration-dependent manner. The general principles underlying the concentration-dependent oligomerization of opsin may be universal and apply to other GPCRs as well.

Stabilization of tryptophan hydroxylase 2 by l‐phenylalanine‐induced dimerization

Tryptophan hydroxylase 2 (TPH2) catalyses the initial and rate‐limiting step in the biosynthesis of serotonin, which is associated with a variety of disorders such as depression, obsessive compulsive disorder, and schizophrenia. Full‐length TPH2 is poorly characterized due to low purification quantities caused by its inherent instability. Three truncated variants of human TPH2 (rch TPH2; regulatory and catalytic domain, NΔ47‐rch TPH2; truncation of 47 residues in the N terminus of rch TPH2, and ch TPH2; catalytic domain) were expressed, purified, and examined for changes in transition temperature, inactivation rate, and oligomeric state. ch TPH2 displayed 14‐ and 11‐fold higher half‐lives compared to rch TPH2 and NΔ47‐rch TPH2, respectively. Differential scanning calorimetry experiments demonstrated that this is caused by premature unfolding of the less stable regulatory domain. By differential scanning fluorimetry, the unfolding transitions of rch TPH2 and NΔ47‐rch TPH2 are found to shift from polyphasic to apparent two‐state by the addition of l‐Trp or l‐Phe. Analytical gel filtration revealed that rch TPH2 and NΔ47‐rch TPH2 reside in a monomer–dimer equilibrium which is significantly shifted toward dimer in the presence of l‐Phe. The dimerizing effect induced by l‐Phe is accompanied by a stabilizing effect, which resulted in a threefold increase in half‐lives of rch TPH2 and NΔ47‐rch TPH2. Addition of l‐Phe to the purification buffer significantly increases the purification yields, which will facilitate characterization of hTPH2.

Activation of Dihydrogen by Masked Doubly Bonded Aluminum Species

AbstractActivation of dihydrogen by masked dialumenes (Al=Al doubly bonded species) is reported. Reactions of barrelene‐type dialumanes, which have the reactivity as masked equivalents of 1,2‐diaryldialumenes ArAl=AlAr, with H2 afforded dihydroalumanes ArAlH2 at room temperature (Ar: bulky aryl groups). These dihydroalumanes form hydrogen‐bridged dimers [ArHAl(μ‐H)]2 in the crystalline state, while a monomer–dimer equilibrium was suggested in solution. The 1,2‐diaryldialumenes generated from the barrelene‐type dialumanes are the putative active species in the cleavage of H2.

The dimerization equilibrium of a ClC Cl(-)/H(+) antiporter in lipid bilayers.

Interactions between membrane protein interfaces in lipid bilayers play an important role in membrane protein folding but quantification of the strength of these interactions has been challenging. Studying dimerization of ClC-type transporters offers a new approach to the problem, as individual subunits adopt a stable and functionally verifiable fold that constrains the system to two states - monomer or dimer. Here, we use single-molecule photobleaching analysis to measure the probability of ClC-ec1 subunit capture into liposomes during extrusion of large, multilamellar membranes. The capture statistics describe a monomer to dimer transition that is dependent on the subunit/lipid mole fraction density and follows an equilibrium dimerization isotherm. This allows for the measurement of the free energy of ClC-ec1 dimerization in lipid bilayers, revealing that it is one of the strongest membrane protein complexes measured so far, and introduces it as new type of dimerization model to investigate the physical forces that drive membrane protein association in membranes.

X-ray Crystallographic Structure and Solution Behavior of an Antiparallel Coiled-Coil Hexamer Formed by de Novo Peptides.

The self-assembly of peptides and proteins into higher-ordered structures is encoded in the amino acid sequence of each peptide or protein. Understanding the relationship among the amino acid sequence, the assembly dynamics, and the structure of well-defined peptide oligomers expands the synthetic toolbox for these structures. Here, we present the X-ray crystallographic structure and solution behavior of de novo peptides that form antiparallel coiled-coil hexamers (ACC-Hex) by an interaction motif neither found in nature nor predicted by existing peptide design software. The 1.70 Å X-ray crystallographic structure of peptide 1a shows six α-helices associating in an antiparallel arrangement around a central axis comprising hydrophobic and aromatic residues. Size-exclusion chromatography studies suggest that peptides 1 form stable oligomers in solution, and circular dichroism experiments show that peptides 1 are stable to relatively high temperatures. Small-angle X-ray scattering studies of the solution behavior of peptide 1a indicate an equilibrium of dimers, hexamers, and larger aggregates in solution. The structures presented here represent a new motif of biomolecular self-assembly not previously observed for de novo peptides and suggest supramolecular design principles for material scaffolds based on coiled-coil motifs containing aromatic residues.

Structural basis for ligand-dependent dimerization of phenylalanine hydroxylase regulatory domain.

The multi-domain enzyme phenylalanine hydroxylase (PAH) catalyzes the hydroxylation of dietary I-phenylalanine (Phe) to I-tyrosine. Inherited mutations that result in PAH enzyme deficiency are the genetic cause of the autosomal recessive disorder phenylketonuria. Phe is the substrate for the PAH active site, but also an allosteric ligand that increases enzyme activity. Phe has been proposed to bind, in addition to the catalytic domain, a site at the PAH N-terminal regulatory domain (PAH-RD), to activate the enzyme via an unclear mechanism. Here we report the crystal structure of human PAH-RD bound with Phe at 1.8 Å resolution, revealing a homodimer of ACT folds with Phe bound at the dimer interface. This work delivers the structural evidence to support previous solution studies that a binding site exists in the RD for Phe, and that Phe binding results in dimerization of PAH-RD. Consistent with our structural observation, a disease-associated PAH mutant impaired in Phe binding disrupts the monomer:dimer equilibrium of PAH-RD. Our data therefore support an emerging model of PAH allosteric regulation, whereby Phe binds to PAH-RD and mediates the dimerization of regulatory modules that would bring about conformational changes to activate the enzyme.

T-to-R switch of muscle fructose-1,6-bisphosphatase involves fundamental changes of secondary and quaternary structure.

Fructose-1,6-bisphosphatase (FBPase) catalyzes the hydrolysis of fructose 1,6-bisphosphate to fructose 6-phosphate and is a key enzyme of gluconeogenesis and glyconeogenesis and, more generally, of the control of energy metabolism and glucose homeostasis. Vertebrates, and notably Homo sapiens, express two FBPase isoforms. The liver isozyme is expressed mainly in gluconeogenic organs, where it functions as a regulator of glucose synthesis. The muscle isoform is expressed in all cells, and recent studies have demonstrated that its role goes far beyond the enzymatic function, as it can interact with various nuclear and mitochondrial proteins. Even in its enzymatic function, the muscle enzyme is different from the liver isoform, as it is 100-fold more susceptible to allosteric inhibition by AMP and this effect can be abrogated by complex formation with aldolase. All FBPases are homotetramers composed of two intimate dimers: the upper dimer and the lower dimer. They oscillate between two conformational states: the inactive T form when in complex with AMP, and the active R form. Parenthetically, it is noted that bacterial FBPases behave somewhat differently, and in the absence of allosteric activators exist in a tetramer-dimer equilibrium even at relatively high concentrations. [Hines et al. (2007), J. Biol. Chem. 282, 11696-11704]. The T-to-R transition is correlated with the conformation of the key loop L2, which in the T form becomes `disengaged' and unable to participate in the catalytic mechanism. The T states of both isoforms are very similar, with a small twist of the upper dimer relative to the lower dimer. It is shown that at variance with the well studied R form of the liver enzyme, which is flat, the R form of the muscle enzyme is diametrically different, with a perpendicular orientation of the upper and lower dimers. The crystal structure of the muscle-isozyme R form shows that in this arrangement of the tetramer completely new protein surfaces are exposed that are most likely targets for the interactions with various cellular and enzymatic partners. The cruciform R structure is stabilized by a novel `leucine lock', which prevents the key residue, Asp187, from locking loop L2 in the disengaged conformation. In addition, the crystal structures of muscle FBPase in the T conformation with and without AMP strongly suggest that the T-to-R transition is a discrete jump rather than a shift of an equilibrium smooth transition through multiple intermediate states. Finally, using snapshots from three crystal structures of human muscle FBPase, it is conclusively demonstrated that the AMP-binding event is correlated with a β→α transition at the N-terminus of the protein and with the formation of a new helical structure.

Examination of ClpB Quaternary Structure and Linkage to Nucleotide Binding

Escherichia coli caseinolytic peptidase B (ClpB) is a molecular chaperone with the unique ability to catalyze protein disaggregation in collaboration with the KJE system of chaperones. Like many AAA+ molecular motors, ClpB assembles into hexameric rings, and this reaction is thermodynamically linked to nucleotide binding. Here we show that ClpB exists in a dynamic equilibrium of monomers, dimers, tetramers, and hexamers in the presence of both limiting and excess ATPγS. We find that ClpB monomer is only able to bind one nucleotide, whereas all 12 sites in the hexameric ring are bound by nucleotide at saturating concentrations. Interestingly, dimers and tetramers exhibit stoichiometries of ∼3 and 7, respectively, which is one fewer than the maximum number of binding sites in the formed oligomer. This observation suggests an open conformation for the intermediates based on the need for an adjacent monomer to fully form the binding pocket. We also report the protein-protein interaction constants for dimers, tetramers, and hexamers and their dependencies on nucleotide. These interaction constants make it possible to predict the concentration of hexamers present and able to bind to cochaperones and polypeptide substrates. Such information is essential for the interpretation of many in vitro studies. Finally, the strategies presented here are broadly applicable to a large number of AAA+ molecular motors that assemble upon nucleotide binding and interact with partner proteins.

Dimerization of hard sphere fluid in inert solvent

Statistical-thermodynamic analysis of the dimerization equilibrium within a hard sphere model in the presence of an inert solvent was carried out. Calculation of the dimerization equilibrium at constant temperature and pressure requires the solution of two equations: the mass action law (MAL) and the equation of state (EOS). Both positive and negative deviations from ideality in a dimerizing hard sphere system with an inert solvent can be simply described by using the minimum number of parameters that characterize the ratio of the components' sizes in the mixture. The enthalpy of mixing varies from the pure endothermic to that of alternating with the exothermic effect at moderate and high concentrations, which are dependent on the ratio of the dimerizing species and the solvent hard-sphere diameters.

Bilayer Modifying Effects of Antipsychotics of Different Generations

Antipsychotics are used to manage the symptoms of schizophrenia (delusions, hallucinations, etc.), to treat bipolar mania, and are commonly used as adjective agents in the treatment of depression. They are known to alter the function of diverse membrane proteins, and for certain antipsychotics (e.g. quintepine), this polypharmacology is thought to be desirable. Because antipsychotics are amphiphiles that modulate the function of different, structurally unrelated membrane proteins, we investigated whether antipsychotics alter lipid bilayer properties at concentrations where they alter membrane protein function. To this end, we used a gramicidin-based fluorescence assay (GBFA) as well as single-channel electrophysiology and we find that antipsychotics like chlorpromazine increase the lifetime and appearance rate of gramicidin (gA) channels, thus shifting the gA monomer dimer equilibrium toward the conducting dimers. We are currently examining other commonly prescribed antipsychotics with the goal of exploring whether there are systematic differences between antipsychotics from the first and second generations. The expectation is that the newer antipsychotics will be less bilayer active and therefore would be expected to have fewer undesired effects. Our other studies have found that an increased alteration of bilayer properties, above a given threshold, may indicate toxicity. Thus, we were keen to learn whether antipsychotics substantially altered bulk bilayer properties and more specifically, we hope to observe whether or not there is a correlation between how well an antipsychotic is tolerated (and thus more commonly prescribed) and its bilayer modifying propensity.

Stripping the CLC-ec1 Dimerization Interface: An Investigation into the Role of Van Der Waals Interactions in Membrane Protein Assembly

What drives membrane proteins to fold and oligomerize in lipid bilayers? One possibility is that protein-protein interactions stabilize the assembled state. In membrane proteins, interfaces are typically lined by non-polar residues offering weak van der Waals interactions (VDW), yet a large network of these might confer strong stability. To investigate the role of side-chain VDW interactions on the free energy of membrane protein complex formation, we have made a library of mutations on the dimerization interface of the CLC-ec1 Cl-/H+ antiporter - a model system that we have developed to measure equilibrium dimerization in lipid bilayers. The interface is comprised of four alpha-helices lined by non-polar residues: I - F219, I220, I223, I227; H - L194, I197, I198, I201; P - L406, I409, I410, L413; Q - I422A, L423A, I426A, I430A, I434A. We systematically mutated each helix surface to alanine (all-ALA), stripping the interface of its VDW interactions. In all cases the protein expresses, is stable and upon reconstitution in membranes, shows Cl- transport activity comparable to wild-type CLC-ec1. We ran gel-filtration chromatography over the course of one week to screen the stoichiometry of the protein in detergent. All-ALA helix I, P & Q are stable dimers, however all-ALA helix H showed shifts to monomer directly after purification. Furthermore, single-mutant L194A was sufficient to shift the protein to a monomer-dimer mixture. Because helix H interacts with helix P, and all-ALA helix P is still dimeric, these results suggest that dimer stability cannot be explained by VDW interactions alone, at least in detergent. We are currently measuring the change in free energy of CLC-ec1 dimerization with these subtractive mutations to quantify the role of VDW interactions in membrane protein assembly.

Measuring Large Membrane Protein Dimerization in Lipid Bilayers by Forster Resonance Energy Transfer

Our understanding of the thermodynamics forces in membrane protein assembly in a lipid bilayer is limited, because there are not many equilibrium models for studying protein association in lipid bilayers. We have developed a new model system based on the homodimeric CLC-ec1 Cl−/H+ antiporter (WT) that offers an opportunity to investigate this. A single tryptophan substitution (I422W) in WT yields a monomer/dimer mixture in detergent. We quantitatively labeled CLC-ec1 with Cy3 donor and/or Cy5 acceptor to measure Forster resonance energy transfer (FRET) changes upon dimerization. By single-molecule TIRF microscopy we measured the molecular FRET efficiency of as 0.7 ± 0.1 (n=3). We then measured the population FRET efficiency of I422W in large membranes while varying the protein/lipid fraction (χ) to determine a Kd of dimerization. Emission spectra obtained from the fluorometer is fit using an in-house software Fytt to decompose contributions from the sensitized emission of donor, acceptor, FRET and background and to quantify FRET efficiency (E). We determined E for the positive dimer control by co-labelling with Cy3/Cy5 across a range of χ. Measuring I422W-Cy3 + I422W-Cy5 in membranes shows low E at low χ that increases and saturates to all-dimer E at high χ, indicating a dimerization reaction in membranes. Furthermore, we observe reversibility of the reaction when going from a high χ to low χ by diluting all-dimer I422W membranes with empty lipid bilayers, and the appearance of FRET upon fusion of separately labelled I422W-Cy3 with I422W-Cy5 membranes, indicating subunit exchange. We compared 2:1 POPE/POPG vs. POPC lipid compositions, and while both show a similar reaction, the POPC data fits well to a dimerization isotherm with Kd(I422W) = 4 x 10−6 protein/lipid, suggesting that we are measuring equilibrium dimerization reaction in membranes.

Biochemical properties of Paracoccus denitrificans FnrP: reactions with molecular oxygen and nitric oxide.

In Paracoccus denitrificans, three CRP/FNR family regulatory proteins, NarR, NnrR and FnrP, control the switch between aerobic and anaerobic (denitrification) respiration. FnrP is a [4Fe–4S] cluster-containing homologue of the archetypal O2 sensor FNR from E. coli and accordingly regulates genes encoding aerobic and anaerobic respiratory enzymes in response to O2, and also NO, availability. Here we show that FnrP undergoes O2-driven [4Fe–4S] to [2Fe–2S] cluster conversion that involves up to 2 O2 per cluster, with significant oxidation of released cluster sulfide to sulfane observed at higher O2 concentrations. The rate of the cluster reaction was found to be ~sixfold lower than that of E. coli FNR, suggesting that FnrP can remain transcriptionally active under microaerobic conditions. This is consistent with a role for FnrP in activating expression of the high O2 affinity cytochrome c oxidase under microaerobic conditions. Cluster conversion resulted in dissociation of the transcriptionally active FnrP dimer into monomers. Therefore, along with E. coli FNR, FnrP belongs to the subset of FNR proteins in which cluster type is correlated with association state. Interestingly, two key charged residues, Arg140 and Asp154, that have been shown to play key roles in the monomer–dimer equilibrium in E. coli FNR are not conserved in FnrP, indicating that different protomer interactions are important for this equilibrium. Finally, the FnrP [4Fe–4S] cluster is shown to undergo reaction with multiple NO molecules, resulting in iron nitrosyl species and dissociation into monomers.Electronic supplementary materialThe online version of this article (doi:10.1007/s00775-015-1326-7) contains supplementary material, which is available to authorized users.

Nuclear magnetic resonance evidence for the dimer formation of beta amyloid peptide 1–42 in 1,1,1,3,3,3-hexafluoro-2-propanol

Alzheimer's disease involves accumulation of senile plaques in which filamentous aggregates of amyloid beta (Aβ) peptides are deposited. Recent studies demonstrate that oligomerization pathways of Aβ peptides may be complicated. To understand the mechanisms of Aβ(1–42) oligomer formation in more detail, we have established a method to produce 15N-labeled Aβ(1–42) suited for nuclear magnetic resonance (NMR) studies. For physicochemical studies, the starting protein material should be solely monomeric and all Aβ aggregates must be removed. Here, we succeeded in fractionating a “precipitation-resistant” fraction of Aβ(1–42) from an “aggregation-prone” fraction by high-performance liquid chromatography (HPLC), even from bacterially overexpressed Aβ(1–42). However, both Aβ(1–42) fractions after 1,1,1,3,3,3-hexafluoro-2-propanol (HFIP) treatment formed amyloid fibrils. This indicates that the “aggregation seed” was not completely monomerized during HFIP treatment. In addition, Aβ(1–42) dissolved in HFIP was found to display a monomer–dimer equilibrium, as shown by two-dimensional 1H–15N NMR. We demonstrated that the initial concentration of Aβ during the HFIP pretreatment altered the kinetic profiles of Aβ fibril formation in a thioflavin T fluorescence assay. The findings described here should ensure reproducible results when studying the Aβ(1–42) peptide.

Measuring Membrane Protein Dimerization Equilibrium in Lipid Bilayers by Single-Molecule Fluorescence Microscopy.

Dimerization of membrane protein interfaces occurs during membrane protein folding and cell receptor signaling. Here, we summarize a method that allows for measurement of equilibrium dimerization reactions of membrane proteins in lipid bilayers, by measuring the Poisson distribution of subunit capture into liposomes by single-molecule photobleaching analysis. This strategy is grounded in the fact that given a comparable labeling efficiency, monomeric or dimeric forms of a membrane protein will give rise to distinctly different photobleaching probability distributions. These methods have been used to verify the dimer stoichiometry of the Fluc F- ion channel and the dimerization equilibrium constant of the ClC-ec1 Cl-/H+ antiporter in lipid bilayers. This approach can be applied to any membrane protein system provided it can be purified, fluorescently labeled in a quantitative manner, and verified to be correctly folded by functional assays, even if the structure is not yet known.