Noncovalent Dimer Research Articles

A Bioorganic Chemistry Approach to Understanding Molecular Recognition in Protein–Nucleic Acid Complexes

Abstract A combination of synthetic, organic, and biochemical approaches has been developed to study how protein and nucleic acid assemblies modulate the affinity, specificity and cooperativity of protein–nucleic acid interactions. These strategies have enabled formation of noncovalent peptide dimers on specific DNA sequences. A new framework from RNA and peptide into a stable complex of ribonucleopeptide was also developed to construct receptors and fluorescent sensors for small molecules. Attempts to design novel DNA-binding peptides, receptors and sensors will provide an ultimate test for our understanding of the principle of molecular recognition associated with protein–nucleic acid interactions.

Removal of a consensus proline is not sufficient to allow tetratricopeptide repeat oligomerization.

Tetratricopeptide repeat (TPR) domains are ubiquitous protein interaction domains that adopt a modular antiparallel array of α-helices. The TPR fold typically adopts a monomeric state, and consensus TPRs sequences successfully fold into the expected monomeric topology. The versatility of the TPR fold also supports different quaternary structures, which may function as regulatory switches. One example is yeast mitochondrial fission 1 (Fis1) that appears to interconvert between monomer and dimer states in regulating division of peroxisomes and mitochondria. Whether human Fis1 can also interconvert like the yeast molecule is unknown. A TPR consensus proline residue present in human Fis1 is absent in the yeast molecule and, when added, prevents yeast Fis1 dimerization suggesting that the TPR consensus proline might have persisted to prevent TPR oligomerization. Here, we address this question with human Fis1 and the consensus TPR protein CTPR3. We demonstrate that human Fis1 does not form a noncovalent dimer via its TPR domain, despite conditions that favor dimerization of the yeast protein. We also show that the presence of the consensus proline is not sufficient to forbid TPR dimerization. Lastly, an analysis of all available TPR protein structures (22 nonredundant structures, totaling 64 TPRs-42 with the consensus proline and 22 without) revealed that the consensus proline is not necessary for turn formation, but does favor shorter turns. This work suggests the TPR consensus proline is not to prevent oligomerization, but to favor tight turns between repeats.

Peripherin-2 and Rom-1 have opposing effects on rod outer segment targeting of retinitis pigmentosa-linked peripherin-2 mutants

Mutations in the photoreceptor outer segment (OS) specific peripherin-2 lead to autosomal dominant retinitis pigmentosa (adRP). By contrast, mutations in the peripherin-2 homolog Rom-1 cause digenic RP in combination with certain heterozygous mutations in peripherin-2. The mechanisms underlying the differential role of peripherin-2 and Rom-1 in RP pathophysiology remained elusive so far. Here, focusing on two adRP-linked peripherin-2 mutants, P210L and C214S, we analyzed the binding characteristics, protein assembly, and rod OS targeting of wild type (perWT), mutant peripherin-2 (perMT), or Rom-1 complexes, which can be formed in patients heterozygous for peripherin-2 mutations. Both mutants are misfolded and lead to decreased binding to perWT and Rom-1. Furthermore, both mutants are preferentially forming non-covalent perMT-perMT, perWT-perMT, and Rom-1-perMT dimers. However, only perWT-perMT, but not perMT-perMT or Rom-1-perMT complexes could be targeted to murine rod OS. Our study provides first evidence that non-covalent perWT-perMT dimers can be targeted to rod OS. Finally, our study unravels unexpected opposing roles of perWT and Rom-1 in rod OS targeting of adRP-linked peripherin-2 mutants and suggests a new treatment strategy for the affected individuals.

Insights into the transmembrane helix associations of kit ligand by molecular dynamics simulation and TOXCAT.

Kit ligand (KITL) plays important roles in cell proliferation, differentiation, and survival via interaction with its receptor Kit. The previous studies demonstrated that KITL formed a noncovalent homodimer through transmembrane (TM) domain; however, the undergoing mechanism of transmembrane association that determines KITL TM dimerization is still not clear. Herein, molecular dynamics (MD) simulation strategy and TOXCAT assay were combined to characterize the dimerization interface and structure of KITL TM in details. KITL TM formed a more energetically favorable noncovalent dimer through a conserved SxxxGxxxG motif in the MD simulation. Furthermore, the TOXCAT results demonstrated that KITL TM self-associated strongly in the bilayer membrane environment. Mutating any one of the small residues Ser11, Gly15 or Gly19 to Ile disrupted KITL TM dimerization dramatically, which further validated our MD simulation results. In addition, our results showed that Tyr22 could help to stabilize the TM interactions via interacting with the phosphoric group in the bilayer membrane. Pro7 did not induce helix kinks or swivel angles in KITL TM, but it was related with the pitch of the turn around this residue so as to affect the dimer formation. Combining the results of computer modeling and experimental mutagenesis studies on the KITL TM provide new insights for the transmembrane helix association of KITL dimerization. Proteins 2017; 85:1362-1370. © 2017 Wiley Periodicals, Inc.

Cytosolic BNIP3 Dimer Interacts with Mitochondrial BAX Forming Heterodimers in the Mitochondrial Outer Membrane under Basal Conditions.

The primary function of mitochondria is energy production, a task of particular importance especially for cells with a high energy demand like cardiomyocytes. The B-cell lymphoma (BCL-2) family member BCL-2 adenovirus E1B 19 kDa-interacting protein 3 (BNIP3) is linked to mitochondrial targeting after homodimerization, where it functions in inner membrane depolarization and permeabilization of the mitochondrial outer membrane (MOM) mediating cell death. We investigated the basal distribution of cardiac BNIP3 in vivo and its physical interaction with the pro-death protein BCL2 associated X, apoptosis regulator (BAX) and with mitochondria using immunoblot analysis, co-immunoprecipitation, and continuous wave and pulsed electron paramagnetic resonance spectroscopy techniques. We found that BNIP3 is present as a dimer in the cytosol and in the outer membrane of cardiac mitochondria under basal conditions. It forms disulfide-bridged, but mainly non-covalent dimers in the cytosol. Heterodimers with BAX are formed exclusively in the MOM. Furthermore, our results suggest that BNIP3 interacts with the MOM directly via mitochondrial BAX. However, the physical interactions with BAX and the MOM did not affect the membrane potential and cell viability. These findings suggest that another stimulus other than the mere existence of the BNIP3/BAX dimer in the MOM is required to promote BNIP3 cell-death activity; this could be a potential disturbance of the BNIP3 distribution homeostasis, namely in the direction of the mitochondria.

Coordination of Sequential Action in Asymmetrical Hexameric ATPase by Arginine Finger

Biomotors are involved in countless vital active processes including muscle motion, heart beating, lung breathing, DNA replication, cell division and viral DNA packaging. In this report, the sequential action of the ATPase ring in the dsDNA packaging motor of phi29 is revealed to be regulated by an arginine finger. Arginine finger is shown to extend from one ATPase subunit to the adjacent one for a noncovalent dimer formation, and is involved in ATP binding, hydrolysis, and DNA translocation. Dimer formation is observed when arginine mutants were mixed with wild-types, which can offer their arginine to promote the inter-subunit interaction. Ultracentrifugation and in vitro virion assembly assays indicated that the ATPase was presenting as monomers and dimer mixtures, based on the results that the isolated dimers alone were deficient in DNA translocation, but the addition of monomer could restore the activity. Moreover, ATP binding or hydrolysis induce two rounds of conformational entropy changes of the ATPase with high or low DNA affinity. Taken together, we concluded that the arginine finger regulates sequential action of the motor ATPase subunit by promoting the formation of the dimer inside the hexamer. The formation of one dimer and four monomers inside the hexamer lead to an asymmetrical configuration of the hexameric ATPase complex. Such organization is supported by structural evidences of many other ATPase systems. All the results above provide clues for why the hexameric phi29 ATPase was previously reported as a pentameric configuration by cryo-electron microscopy (cryo-EM). Since the bridging by the arginine finger renders two adjacent ATPase subunits closer than other subunits, thus, the asymmetrical hexamer would appear as a pseudo-pentamer by cryo-EM, a technology that acquires the average of numerous images.

Conformational behavior and stacking interactions of contorted polycyclic aromatics

We present a systematic computational analysis of the conformations and stacking interactions of a set of 18 saddle-shaped, contorted polycyclic aromatic compounds at the B97-D3M(BJ)/TZV(2d,2p)//B97-D/TZV(2d,2p) level of theory. These doubly-concave systems offer a means of tuning the strength of stacking interactions through variations in molecular curvature, and understanding the intermolecular non-covalent interactions exhibited by these systems will aid the design of contorted polycyclic systems with precisely defined packing in the solid state. Computations reveal wide variations in both the nature of the low-lying conformations and the stacking affinities of these systems. In particular, the introduction of both thiophene rings around the periphery of these systems and the incorporation of B and N atoms into the coronene core can greatly enhance their tendency to form strongly stacked dimers. Overall, these data provide a reminder that curvature does not always lead to stronger stacking interactions.

Defining Noncovalent Ubiquitin Homodimer Interfacial Interactions through Comparisons with Covalently Linked Diubiquitin

Covalently linked diubiquitin (diUbq) is known to adopt specific interfacial interactions owing to steric hindrance induced by the covalent tether. K48-linked diUbq preferentially forms hydrophobic interfacial interactions between the two I44 faces under physiological conditions, whereas K63-linked diUbq preferentially forms electrostatic interfacial interactions. Here, we show using collision-induced unfolding ion mobility-mass spectrometry that the recently reported noncovalent dimer of ubiquitin exhibits structural preferences and interfacial interactions that are most similar to that of K48-linked diUbq.

The Monomer Electron Density Force Field (MEDFF): A Physically Inspired Model for Noncovalent Interactions.

We propose a methodology to derive pairwise-additive noncovalent force fields from monomer electron densities without any empirical input. Energy expressions are based on the symmetry-adapted perturbation theory (SAPT) decomposition of interaction energies. This ensures a physically motivated force field featuring an electrostatic, exchange-repulsion, dispersion, and induction contribution, which contains two types of parameters. First, each contribution depends on several fixed atomic parameters, resulting from a partitioning of the monomer electron density. Second, each of the last three contributions (exchange-repulsion, dispersion, and induction) contains exactly one linear fitting parameter. These three so-called interaction parameters in the model are initially estimated separately using SAPT reference calculations for the S66x8 database of noncovalent dimers. In a second step, the three interaction parameters are further refined simultaneously to reproduce CCSD(T)/CBS interaction energies for the same database. The limited number of parameters that are fitted to dimer interaction energies (only three) avoids ill-conditioned fits that plague conventional parameter optimizations. For the exchange-repulsion and dispersion component, good results are obtained for all dimers in the S66x8 database using one single value for the associated interaction parameters. The values of those parameters can be considered universal and can also be used for dimers not present in the original database used for fitting. For the induction component such an approach is only viable for the dispersion-dominated dimers in the S66x8 database. For other dimers (such as hydrogen-bonded complexes), we show that our methodology remains applicable. However, the interaction parameter needs to be determined on a case-specific basis. As an external validation, the force field predicts interaction energies in good agreement with CCSD(T)/CBS values for dispersion-dominated dimers extracted from an HIV-II protease crystal structure with a bound ligand (indinavir). Furthermore, experimental second virial coefficients of small alkanes and alkenes are well reproduced.

P-Hydroxyphenyl-pyranoanthocyanins: An Experimental and Theoretical Investigation of Their Acid-Base Properties and Molecular Interactions.

The physicochemical properties of the wine pigments catechyl-pyranomalvidin-3-O-glucoside (PA1) and guaiacyl-pyranomalvidin-3-O-glucoside (PA2) are extensively revisited using ultraviolet (UV)-visible spectroscopy, dynamic light scattering (DLS) and quantum chemistry density functional theory (DFT) calculations. In mildly acidic aqueous solution, each cationic pigment undergoes regioselective deprotonation to form a single neutral quinonoid base and water addition appears negligible. Above pH = 4, both PA1 and PA2 become prone to aggregation, which is manifested by the slow build-up of broad absorption bands at longer wavelengths (λ ≥ 600 nm), followed in the case of PA2 by precipitation. Some phenolic copigments are able to inhibit aggregation of pyranoanthocyanins (PAs), although at large copigment/PA molar ratios. Thus, chlorogenic acid can dissociate PA1 aggregates while catechin is inactive. With PA2, both chlorogenic acid and catechin are able to prevent precipitation but not self-association. Calculations confirmed that the noncovalent dimerization of PAs is stronger with the neutral base than with the cation and also stronger than π–π stacking of PAs to chlorogenic acid (copigmentation). For each type of complex, the most stable conformation could be obtained. Finally, PA1 can also bind hard metal ions such as Al3+ and Fe3+ and the corresponding chelates are less prone to self-association.

Construction of Asymmetrical Hexameric Biomimetic Motors with Continuous Single-Directional Motion by Sequential Coordination.

The significance of bionanomotors in nanotechnology is analogous to mechanical motors in daily life. Here the principle and approach for designing and constructing biomimetic nanomotors with continuous single-directional motion are reported. This bionanomotor is composed of a dodecameric protein channel, a six-pRNA ring, and an ATPase hexamer. Based on recent elucidations of the one-way revolving mechanisms of the phi29 double-stranded DNA (dsDNA) motor, various RNA and protein elements are designed and tested by single-molecule imaging and biochemical assays, with which the motor with active components has been constructed. The motor motion direction is controlled by three operation elements: (1) Asymmetrical ATPase with ATP-interacting domains for alternative DNA binding/pushing regulated by an arginine finger in a sequential action manner. The arginine finger bridges two adjacent ATPase subunits into a non-covalent dimer, resulting in an asymmetrical hexameric complex containing one dimer and four monomers. (2) The dsDNA translocation channel as a one-way valve. (3) The hexameric pRNA ring geared with left-/right-handed loops. Assessments of these constructs reveal that one inactive subunit of pRNA/ATPase is sufficient to completely block motor function (defined as K = 1), implying that these components work sequentially based on the principle of binomial distribution and Yang Hui's triangle.

An Arginine Finger Regulates the Sequential Action of Asymmetrical Hexameric ATPase in the Double-Stranded DNA Translocation Motor.

Biological motors are ubiquitous in living systems. Currently, how the motor components coordinate the unidirectional motion is elusive in most cases. Here, we report that the sequential action of the ATPase ring in the DNA packaging motor of bacteriophage ϕ29 is regulated by an arginine finger that extends from one ATPase subunit to the adjacent unit to promote noncovalent dimer formation. Mutation of the arginine finger resulted in the interruption of ATPase oligomerization, ATP binding/hydrolysis, and DNA translocation. Dimer formation reappeared when arginine mutants were mixed with other ATPase subunits that can offer the arginine to promote their interaction. Ultracentrifugation and virion assembly assays indicated that the ATPase was presenting as monomers and dimer mixtures. The isolated dimer alone was inactive in DNA translocation, but the addition of monomer could restore the activity, suggesting that the hexameric ATPase ring contained both dimer and monomers. Moreover, ATP binding or hydrolysis resulted in conformation and entropy changes of the ATPase with high or low DNA affinity. Taking these observations together, we concluded that the arginine finger regulates sequential action of the motor ATPase subunit by promoting the formation of the dimer inside the hexamer. The finding of asymmetrical hexameric organization is supported by structural evidence of many other ATPase systems showing the presence of one noncovalent dimer and four monomer subunits. All of these provide clues for why the asymmetrical hexameric ATPase gp16 of ϕ29 was previously reported as a pentameric configuration by cryo-electron microscopy (cryo-EM) since the contact by the arginine finger renders two adjacent ATPase subunits closer than other subunits. Thus, the asymmetrical hexamer would appear as a pentamer by cryo-EM, a technology that acquires the average of many images.

How Accurate Are the Minnesota Density Functionals for Noncovalent Interactions, Isomerization Energies, Thermochemistry, and Barrier Heights Involving Molecules Composed of Main-Group Elements?

The 14 Minnesota density functionals published between the years 2005 and early 2016 are benchmarked on a comprehensive database of 4986 data points (84 data sets) involving molecules composed of main-group elements. The database includes noncovalent interactions, isomerization energies, thermochemistry, and barrier heights, as well as equilibrium bond lengths and equilibrium binding energies of noncovalent dimers. Additionally, the sensitivity of the Minnesota density functionals to the choice of basis set and integration grid is explored for both noncovalent interactions and thermochemistry. Overall, the main strength of the hybrid Minnesota density functionals is that the best ones provide very good performance for thermochemistry (e.g., M06-2X), barrier heights (e.g., M08-HX, M08-SO, MN15), and systems heavily characterized by self-interaction error (e.g., M06-2X, M08-HX, M08-SO, MN15), while the main weakness is that none of them are state-of-the-art for the full spectrum of noncovalent interactions and isomerization energies (although M06-2X is recommended from the 10 hybrid Minnesota functionals). Similarly, the main strength of the local Minnesota density functionals is that the best ones provide very good performance for thermochemistry (e.g., MN15-L), barrier heights (e.g., MN12-L), and systems heavily characterized by self-interaction error (e.g., MN12-L and MN15-L), while the main weakness is that none of them are state-of-the-art for the full spectrum of noncovalent interactions and isomerization energies (although M06-L is clearly the best from the four local Minnesota functionals). As an overall guide, M06-2X and MN15 are perhaps the most broadly useful hybrid Minnesota functionals, while M06-L and MN15-L are perhaps the most broadly useful local Minnesota functionals, although each has different strengths and weaknesses.

Big Changes for Small Noncovalent Dimers: Revisiting the Potential Energy Surfaces of (P2)2 and (PCCP)2 with CCSD(T) Optimizations and Vibrational Frequencies.

This article details the re-examination of low-lying stationary points on the potential energy surfaces (PESs) of two challenging noncovalent homogeneous dimers, (P2)2 and (PCCP)2. The work was motivated by the rather large differences between MP2 and CCSD(T) energetics that were recently reported for these systems (J. Comput. Chem. 2014, 35, 479-487). The current investigation reveals significant qualitative and quantitative changes when the CCSD(T) method is used to characterize the stationary points instead of MP2. For example, CCSD(T) optimizations and harmonic vibrational frequency computations with the aug-cc-pVTZ basis set indicate that the parallel-slipped (PS) structure is the only P2 dimer stationary point examined that is a minimum (zero imaginary frequencies, ni = 0), whereas prior MP2 computations indicated that it was a transition state (ni = 1). Furthermore, the L-shaped structure of (P2)2 was the only minimum according to MP2 computations, but it collapses to the PS structure on the CCSD(T)/aug-cc-pVTZ PES. For the larger PCCP dimer, the CCSD(T) computations reveal that four rather than just two of the six stationary points characterized are minima. A series of explicitly correlated single-point energies were computed for all of the optimized structures to estimate the MP2 and CCSD(T) electronic energies at the complete basis set limit. CCSDT(Q) computations were also performed to assess the effects of dynamical electron correlation beyond the CCSD(T) level. For both (P2)2 and (PCCP)2, dispersion remains the dominant attractive component to the interaction energy according to symmetry-adapted perturbation theory analyses, and it is also the most challenging component to accurately evaluate.

Ligand-dependent responses of the silkworm prothoracicotropic hormone receptor, Torso, are maintained by unusual intermolecular disulfide bridges in the transmembrane region.

The insect membrane-protein, Torso, is a member of the receptor-tyrosine-kinase family, and is activated by its ligand, prothoracicotropic hormone (PTTH). Although PTTH is one of the most important regulators of insect development, the mechanism of Torso activation by the hormone has remained elusive. In this study, using heterologous expression in cultured Drosophila S2 cells, we detected ligand-independent dimerization of silkworm Torso, and found that the receptor molecules in the dimer were linked by intermolecular disulfide bridges. By examining the oligomerization states of several truncation and substitution mutants of Torso, atypical cysteine residues in the transmembrane region were identified as being responsible for the intermolecular linkage in the dimer. The replacement of all of the cysteines in the region with phenylalanines abolished the disulfide-bond-mediated dimerization; however, non-covalent dimerization of the mutant was detected using a cross-linking reagent, both with and without ligand stimulation. This non-covalent dimerization caused apparent receptor autophosphorylation independently of the ligand stimulation, but did not promote the ERK phosphorylation in the downstream signaling pathway. The unique Torso structure with the intermolecular disulfide bridges in the transmembrane region is necessary to maintain the ligand-dependent receptor functions of autophosphorylation and downstream activation.

Structure, Aggregation, and Activity of a Covalent Insulin Dimer Formed During Storage of Neutral Formulation of Human Insulin

A specific covalently linked dimeric species of insulin high molecular weight products (HMWPs), formed during prolonged incubation of a neutral pharmaceutical formulation of human insulin, were characterized in terms of tertiary structure, self-association, biological activity, and fibrillation properties. The dimer was formed by a covalent link between A21Asn and B29Lys. It was analyzed using static and dynamic light scattering and small-angle X-ray scattering to evaluate its self-association behavior. The tertiary structure was obtained using nuclear magnetic resonance and X-ray crystallography. The biological activity of HMWP was determined using 2 in vitro assays, and its influence on fibrillation was investigated using Thioflavin T assays. The dimer's tertiary structure was nearly identical to that of the noncovalent insulin dimer, and it was able to form hexamers in the presence of zinc. The dimer exhibited reduced propensity for self-association in the absence of zinc but significantly postponed the onset of fibrillation in insulin formulations. Consistent with its dimeric state, the tested species of HMWP showed little to no biological activity in the used assays. This study is the first detailed characterization of a specific type of human insulin HMWP formed during storage of a marketed pharmaceutical formulation. These results indicate that this specific type of HMWP is unlikely to antagonize the physical stability of the formulation, as HMWP retained a tertiary structure similar to the noncovalent dimer and participated in hexamer assembly in the presence of zinc. In addition, increasing amounts of HMWP reduce the rate of insulin fibrillation.

The Influence of Alkali Metal Cation Size on the Formation and Dissociation of Crown Ether Fullerene Dimers in Electrospray Mass Spectrometry

Crown ether fullerenes, C60H(cr – H), form two different dimers in electrospray ionization mass spectrometry, a noncovalently bound, metal-bridged dimer [C60H(cr – H)]2M+, and a singly bonded fullerene dimer of the type (cr – H)–C60–C60–(cr – H)Mnn+, with n = 1, 2. In this work, the dependence of both the formation and the dissociation of these dimers on the respective sizes of the crown ether moiety (12cr4, 15cr5, 18cr6) and of the alkali metal ion (M+ = Li+, Na+, K+, Rb+, Cs+) is studied. The size ratio of crown ether cavity to metal cation size has a profound influence on the formation of the noncovalent dimers, [C60H(cr – H)]2M+. These are only formed if the cation is larger than the crown ether cavity. Also, the doubly charged, covalent dimers [C60(cr – H)]2M22+ show a dependence on the size ratio. The proposed formation mechanism involves the recombination of two C60(cr – H)M•+ radical cations, which can only occur if the repulsion between the two charges is sufficiently reduced by encapsulating the...

Water-Mediated Dimerization of Ubiquitin Ions Captured by Cryogenic Ion Mobility-Mass Spectrometry.

The dynamics, structures, and functions of most biological molecules are strongly influenced by the nature of the peptide's or protein's interaction with water. Here, cryogenic ion mobility-mass spectrometry studies of ubiquitin have directly captured a water-mediated protein-protein binding event involving hydrated, noncovalently bound dimer ions in solution, and this interaction has potential relevance to one of the most important protein-protein interactions in nature. As solvent is removed, dimer ions, viz. [2 M + 14H](14+), can be stabilized by only a few attached water molecules prior to dissociation into individual monomeric ions. The hydrophobic patch of ubiquitin formed by the side chains of Leu-8, Ile-44, and Val-70 meet all the necessary conditions for a protein-protein binding "hot spot," including the requirement for occlusion of water to nearby hydrophilic sites, and it is suggested that this interaction is responsible for formation of the hydrated noncovalent ubiquitin dimer.

Mapping the heparin-binding site of the BMP antagonist gremlin by site-directed mutagenesis based on predictive modelling.

Gremlin is a member of the CAN (cerberus and DAN) family of secreted BMP (bone morphogenetic protein) antagonists and also an agonist of VEGF (vascular endothelial growth factor) receptor-2. It is critical in limb skeleton and kidney development and is re-expressed during tissue fibrosis. Gremlin binds strongly to heparin and heparan sulfate and, in the present study, we sought to investigate its heparin-binding site. In order to explore a putative non-contiguous binding site predicted by computational molecular modelling, we substituted a total of 11 key arginines and lysines located in three basic residue sequence clusters with homologous sequences from cerberus and DAN (differential screening selected gene abberative in neuroblastoma), CAN proteins which lack basic residues in these positions. A panel of six Myc-tagged gremlin mutants, MGR-1-MGR-6 (MGR, mutant gremlin), each containing different combinations of targeted substitutions, all showed markedly reduced affinity for heparin as demonstrated by their NaCl elution on heparin affinity chromatography, thus verifying our predictions. Both MGR-5 and MGR-6 retained BMP-4-binding activity comparable to that of wild-type gremlin. Low-molecular-mass heparin neither promoted nor inhibited BMP-4 binding. Finally, glutaraldehyde cross-linking demonstrated that gremlin forms non-covalent dimers, similar behaviour to that of DAN and also PRDC (protein related to cerberus and DAN), another CAN protein. The resulting dimer would possess two heparin-binding sites, each running along an exposed surface on the second β-strand finger loop of one of the monomers.

Effect of Self-Association on the Phase Stability of Triphenylamine Derivatives.

The self-association equilibrium, i.e. formation of noncovalent dimers, in two triphenylamine derivatives, TPD (N,N'-bis(3-methylphenyl)-N,N'-diphenylbenzidine) and mMTDAB (1,3,5-tris[(3-methylphenyl)phenylamino]benzene), in solution was evaluated by (1)H NMR spectroscopy. The gas-phase energetics of the respective dimerization processes was explored by computational quantum chemistry. The results indicate that self-association is significantly more extensive in TPB than in TDAB. It is proposed that this fact helps to explain why TPB presents a stability higher than expected in the liquid phase, which is reflected in a lower melting temperature, a less volatile liquid, and possibly a higher tendency to form a glass. These results highlight the influence of self-association on the phase equilibria and thermodynamic properties of pure organic substances.