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Hydrogen cyanide polymerizes readily under a variety of conditions and significant prebiotic roles have been suggested for these polymers due to the abundance of HCN in universe. However, the structures of HCN polymers have been more speculative than grounded in experimental data. Here we show that 13C and 15N solid state NMR spectra of polymers formed in neat HCN are inconsistent with the previously proposed structures and suggest instead that the polymers are formed by simple monomer addition, first in head-to-tail fashion to form linear, conjugated chains, and then laterally to form saturated two-dimensional networks. This interpretation of the NMR spectra finds support in other information about the polymerization of neat HCN, including the presence of free radicals. As expected from the literature, formation of the HCN tetramer, diaminomaleonitrile, is also observed, but only when the reaction is catalyzed exclusively by base and then in crystalline form. [ABSTRACT FROM AUTHOR]

Cyclodextrins (CDs) are cyclic oligomers of a-1,4-D-glucopyranoside and are known mainly as hexamers to octamers. The central cavities of CDs can retain small molecules, enabling diverse applications. The smallest members, CD3 and CD4, have ring sizes too small to permit the most stable conformations of glucopyranose and have not been accessible synthetically. In this study, we present methods to chemically synthesize both CD3 and CD4. The main factor in the successful synthesis is the creation of a glucopyranose ring conformationally counterbalanced between equatorial- and axial-rich forms. This suppleness is imparted by a bridge between O-3 and O-6 of glucose, which enables the generation of desirable, albeit deformed, conformers when synthesizing the cyclic trimer and tetramer. [ABSTRACT FROM AUTHOR]

Ab initio calculations predict the existence of the dimer and tetramer of HXeCCH. The interaction energies are -6.66 and -19.40 kcal mol-1 for the dimer and tetramer, respectively. For both complexes, larger blue shifts of the Xe–H stretching mode are found, while the Xe–C stretching modes are slightly redshifted. The stability and structure of HXeCCH crystals is predicted by density functional theory calculations with periodic boundary conditions. Strong electrostatic interactions are found between the monomers in the crystal. The results are first evidence for the existence of crystalline materials made of a novel class of noble gas molecules. [ABSTRACT FROM AUTHOR]

We present an investigation of the thermal dissociation of the gas phase, non-covalent streptavidin tetramer ion. Using an extended desolvating capillary, it was observed that this complex fragments at an activation energy around 75 kJ/mole. The fragmentation occurs via charge migration and the ejection of a single monomer that carries half the total charge. Temperature increases result in intensive supermetallization by sodium ions and, eventually, in thermal dissociation. Using the in-ESI source H/D exchange approach, we observed that deuterium scrambling during the dissociation is relatively low. To better understand the charged droplets dynamics, we performed accurate computer simulations of airflow inside the desolvating capillary, including calculating the gas velocity, temperature and pressure distributions. [ABSTRACT FROM AUTHOR]

Abstract: Although it is universally accepted that dopamine transporters (DATs) exist in monomers, dimers and tetramers (i.e. dimers of dimers), it is not known whether the oligomeric organization of DAT is a prerequisite for its ability to take up dopamine (DA), or whether each DAT protomer, the subunit of quaternary structure, functions independently in terms of DA translocation. In this study, copper phenanthroline (CuP) was used to selectively target surface DAT: increasing concentrations of CuP gradually cross‐linked natural DAT dimers in LLC‐PK1 cells stably expressing hDAT and thereby reduced DA uptake functionality until all surface DATs were inactivated. DATs that were not cross‐linked by CuP showed normal DA uptake with DA Km at ~ 0.5 μM and DA efflux with basal and amphetamine‐induced DA efflux as much as control values. The cocaine analog 2β‐carbomethoxy‐3β‐[4‐fluorophenyl]‐tropane (CFT) was capable to bind to copper‐cross‐linked DATs, albeit with an affinity more than fivefold decreased (Kd of CFT = 109 nM after cross‐linking vs 19 nM before). A kinetic analysis is offered describing the changing amounts of dimers and monomers with increasing [CuP], allowing the estimation of dimer functional activity compared with a DAT monomer. Consonant with previous conclusions for serotonin transporter and NET that only one protomer of an oligomer is active at the time, the present data indicated a functional activity of the DAT dimer of 0.74 relative to a monomer. [ABSTRACT FROM AUTHOR]

Herein, A549 tumor cell proliferation was confirmed to be positively dependent on the concentration of Fe3+ or transferrin (Tf). Gd@C82(OH)22 or C60(OH)22 effectively inhibited the iron uptake and the subsequent proliferation of A549 cells. The conformational changes of Tf mixed with FeCl3, GdCl3, C60(OH)22 or Gd@C82(OH)22 were obtained by SAXS. The results demonstrate that Tf homodimers can be decomposed into monomers in the presence of FeCl3, GdCl3 or C60(OH)22, but associated into tetramers in the presence of Gd@C82(OH)22. The larger change of SAXS shapes between Tf+C60(OH)22 and Tf+FeCl3 implies that C60(OH)22 is bound to Tf, blocking the iron-binding site. The larger deviation of the SAXS shape from a possible crystal structure of Tf tetramer implies that Gd@C82(OH)22 is bound to the Tf tetramer, thus disturbing iron transport. This study well explains the inhibition mechanism of Gd@C82(OH)22 and C60(OH)22 on the iron uptake and the proliferation of A549 tumor cells and highlights the specific interactions of a nanomedicine with the target biomolecules in cancer therapy. [ABSTRACT FROM AUTHOR]

It is known that melittin in an aqueous solution undergoes a conformational transition between the monomer and tetramer by variation in temperature. The transition correlates closely with isomers of the proline residue; monomeric melittin including a trans proline peptide bond ( trans-monomer) is involved directly in the transition, whereas monomeric melittin having a cis proline peptide bond ( cis-monomer) is virtually not. The transition has been explored by using nuclear magnetic resonance spectroscopy in order to clarify the stability of the tetrameric conformation and the cooperativity of the transition. In the light of temperature dependence of chemical shifts of resonances from the isomeric monomers, we qualitatively estimate the temperature-, salt-, and concentration-dependence of the relative equilibrium populations of the trans-monomer and tetramer, and show that the tetramer has a maximum conformational stability at 30-45 °C and that the transition cooperativity is very low. [ABSTRACT FROM AUTHOR]

The ability to measure the thermodynamic stability of proteins with precision is important for both academic and applied research. Such measurements rely on mathematical models of the protein denaturation profile, i.e. the relation between a global protein signal, corresponding to the folding states in equilibrium, and the variable value of a denaturing agent, either heat or a chemical molecule, e.g. urea or guanidinium hydrochloride. In turn, such models rely on a handful of physical laws: the laws of mass action and conservation, the law that relates the protein signal and concentration, and the one that relates stability and denaturant value. So far, equations have been derived mainly for the denaturation profiles of homomeric proteins. Here, we review the underlying basic physical laws and show in detail how to derive model equations for the unfolding equilibria of homomeric or heteromeric proteins up to trimers and potentially tetramers, with or without folding intermediates, and give full demonstrations. We show that such equations cannot be derived for pentamers or higher oligomers except in special degenerate cases. We expand the method to signals that do not correspond to extensive protein properties. We review and expand methods for uncovering hidden intermediates of unfolding. Finally, we review methods for comparing and interpreting the thermodynamic parameters that derive from stability measurements for cognate wild-type and mutant proteins. This work should provide a robust theoretical basis for measuring the stability of complex proteins. [ABSTRACT FROM AUTHOR]

Pyropheophorbides, chlorophyll- a derivatives, were quadruply esterified at the 17-propionate residue with a tetraol to give the covalently-linked tetramers. The synthetic tetramers were mixed with a C 70 fullerene derivative for utilization as the active layer in bulk heterojunction organic solar cells. Their photovoltaic performances were comparable to those of the corresponding monomeric pigments. The similar power conversion efficiencies would be ascribable to the increase of carrier mobility in the tetramers and the partial suppression of photoexcited state of the chlorin π-systems by intramolecular quenching. Photophysical properties of synthetic covalently-linked mesopyropheophorbide- a dimer, trimer, and tetramer as well as its monomer in a solution supported the intramolecular interaction of chlorin moieties to quench their singlet excited states, which was especially observed in the trimer and tetramer. [ABSTRACT FROM AUTHOR]

G-Protein-coupled receptors (GPCRs) were classically described as monomers. We now appreciate that they also function as homo- and hetero-oligomers, for which structural information is lacking. Here, we use available 3D structures and biochemical considerations to present and evaluate experimentally testable structural models for GPCR oligomers and associated G proteins. [ABSTRACT FROM AUTHOR]

The molecular machinery of the cyanobacterial circadian clock oscillator consists of three proteins, KaiA, KaiB and KaiC, which interact with each other to generate circadian oscillations in the presence of ATP (the in vitro Kai ABC clock oscillator). KaiB comprises four subunits organized as a dimer of dimers. Our previous study suggested that, on interaction with KaiC, the tetrameric KaiB molecule dissociates into two molecules of dimeric KaiB. It is uncertain whether KaiB also exists as a monomer and whether the KaiB monomer can drive normal circadian oscillation. To address these questions, we constructed a new KaiB oligomer mutant with an N-terminal deletion, KaiB10-108. KaiB10-108 was a monomer at 4 °C but a dimer at 35 °C. KaiB10-108 was able to drive normal clock oscillation in an in vitro reconstituted Kai ABC clock oscillator at 25 °C, but it was not able to drive normal circadian gene expression rhythms in cyanobacterial cells at 41 °C. Wild-type KaiB existed in equilibrium between a dimer and tetramer at lower KaiB concentrations or in the presence of 1 m NaCl. Our findings suggest that KaiB is in equilibrium between a monomer, dimer and tetramer in cyanobacterial cells. [ABSTRACT FROM AUTHOR]

G protein-coupled receptors can be reconstituted as monomers in nanodiscs and as tetramers in liposomes. When reconstituted with G proteins, both forms enable an allosteric interaction between agonists and guanylyl nucleotides. Both forms, therefore, are candidates for the complex that controls signaling at the level of the receptor. To identify the biologically relevant form, reconstituted monomers and tetramers of the purified M2 muscarinic receptor were compared with muscarinic receptors in sarcolemmal membranes for the effect of guanosine 5'-[β,γ- imido]triphosphate (GMP-PNP) on the inhibition of N-[3H]- methylscopolamine by the agonist oxotremorine-M. With monomers, a stepwise increase in the concentration of GMPPNP effected a lateral, rightward shift in the semilogarithmic binding profile (i.e. a progressive decrease in the apparent affinity of oxotremorine-M). With tetramers and receptors in sarcolemmal membranes, GMP-PNP effected a vertical, upward shift (i.e. an apparent redistribution of sites from a state of high affinity to one of low affinity with no change in affinity per se). The data were analyzed in terms of a mechanistic scheme based on a ligand-regulated equilibrium between uncoupled and G protein-coupled receptors (the "ternary complex model"). The model predicts a rightward shift in the presence of GMP-PNP and could not account for the effects at tetramers in vesicles or receptors in sarcolemmal membranes. Monomers present a special case of the model in which agonists and guanylyl nucleotides interact within a complex that is both constitutive and stable. The results favor oligomers of the M2 receptor over monomers as the biologically relevant state for coupling to G proteins. [ABSTRACT FROM AUTHOR]

Abstract: Nucleoside diphosphate kinase from a moderate halophile Halomonas sp. 593 (HaNDK) is dimer, while NDK from different origins has been shown to assemble into hexamer, or tetramer. Similar to HaNDK, halophilic NDK from Chromohalobacter salexigens DSM3043 (CsNDK) formed dimeric structure. Cysteine139 conserved between HaNDK and CsNDK is located in the monomer/monomer interface. Substitution of Cys139 for Ser caused dissociation of dimeric CsNDK into monomer in Tris buffer, as determined by field flow fractionation technique. Circular dichroism (CD) profile of the mutant CsNDK was nearly identical to the wild type CsNDK: however, the mutant CsNDK became more susceptible to “endproteinase GluC” cleavage, which could be suppressed by an NDK substrate, ATP. The monomer was enzymatically active, although it is possible that active structure is dimer in the presence of substrate. [Copyright &y& Elsevier]

α2-Macroglobulin (α2M) is a broad spectrum proteinase inhibitor that when activated by proteinases (α2M*) undergoes a major conformational change exposing receptor recognition sites in each of its four subunits. These complexes bind to two distinct receptors, namely, the low-density lipoprotein receptor-related protein (LRP) and cell surface glucose-regulated protein [Mr ∼78000 (GRP78)]. The latter is a very high affinity receptor (Kd = 50-100 pM) whose ligation triggers pro-proliferative and anti-apoptotic signaling cascades. Despite its four binding sites, Scatchard analysis of binding of α2M* to cells does not yield a cooperative plot. We, therefore, hypothesize that a monomeric cloned and expressed α2M receptor binding domain (RBD) should trigger comparable signaling events. Indeed, RBD or its K1370A mutant that binds to GRP78 but cannot bind to LRP regulates DNA and protein synthesis by human prostate cancer cells in a manner comparable to that of α2M*. Akt and mTORC1 activation and signaling are also comparably upregulated by α2M*, RBD, or mutant K1370A. Antibodies directed against the carboxyl-terminal domain of GRP78 are antagonists that block α2M*-mediated effects on pro-proliferative and anti-apoptotic signaling cascades and protein and DNA synthesis. The effects of RBD and its mutant were similarly blocked by these antibodies. Finally, proteolysis of α2M at pH values from 5.7 to 7.0 causes production of free RBD and RBD-containing fragments. Thus, while α2M* ligates only one GRP78 receptor molecule per α2M*, it may potentially serve as a reservoir for release of up to four binding fragments per molecule. [ABSTRACT FROM AUTHOR]

The 84-residue homotetrameric BBAT1 is one of the smallest stable protein complexes and therefore is a good test system to study the self-assembly of multimeric proteins. We have researched for this protein the interplay between the folding of monomers and their assembly into tetramers. Replica exchange molecular dynamics simulations relying on a Go model are compared with earlier simulations that use the physics-based coarse-grained UNRES model. [ABSTRACT FROM AUTHOR]

Photoferrotrophy is presumed to be an ancient type of photosynthetic metabolism in which bacteria use the reducing power of ferrous iron to drive carbon fixation. In this work the putative iron oxidoreductase of the photoferrotroph Rhodobacter ferrooxidans SW2 was cloned, purified, and characterized for the first time. This protein, FoxE, was characterized using spectroscopic, thermodynamic, and kinetic techniques. It is a c-type cytochrome that forms a trimer or tetramer in solution; the two hemes of each monomer are hexacoordinated by histidine and methionine. The hemes have positive reduction potentials that allow downhill electron transfer from many geochemically relevant ferrous iron forms to the photosynthetic reaction center. The reduction potentials of the hemes are different and are cross-assigned to fast and slow kinetic phases of ferrous iron oxidation in vitro. Lower reactivity was observed at high pH and may contribute to prevent ferric iron precipitation inside or at the surface of the cell. These results help fill in the molecular details of a metabolic process that likely contributed to the deposition of precambrian banded iron formations, globally important sedimentary rocks that are found on every continent today. [ABSTRACT FROM AUTHOR]

Fluorescence correlation spectroscopy (FCS) and photon counting histogram (PCH) are techniques with single molecule sensitivity that are well suited for examining the biophysical properties of protein complexes in living cells. In the present study, FCS and PCH were applied to determine the diffusion coefficient and oligomeric size of G-protein-coupled receptors. FCS was used to record fluctuations in fluorescence intensity arising from fluorescence-tagged 5-hydroxytryptamine 2C (5-HT2C) receptors diffusing within the plasma membrane of HEK293 cells and rat hippocampal neurons. Autocorrelation analysis yielded diffusion coefficients ranging from 0.8 to 1.2 μm2/s for fluorescence-tagged receptors. Because the molecular brightness of a fluorescent protein is directly proportional to the number of fluorescent proteins traveling together within a protein complex, it can be used to determine the oligomeric size of the protein complex. FCS and PCH analysis of fluorescencetagged 5-HT2C receptors provided molecular brightness values that were twice that of GFP and YFP monomeric controls, similar to a dimeric GFP control, and unaltered by 5-HT. Bimolecular fluorescence complementation of the N- and C-terminal halves of YFP attached to 5-HT2C receptors was observed in endoplasmic reticulum/Golgi and plasma membranes with a brightness equal to monomeric YFP. When GFP-tagged 5-HT2C receptors were co-expressed with a large excess of untagged, non-fluorescent 5-HT2C receptors, the molecular brightness was reduced by half. PCH analysis of the FCS data were best described by a one-component dimer model without monomers or tetramers. Therefore, it is concluded that 5-HT2C receptors freely diffusing within the plasma membrane are dimeric. [ABSTRACT FROM AUTHOR]

Melittin, a peptide of 26 amino acid residues, has been used as a model peptide for protein folding and unfolding, and extensive research has been done into its structure and conformational stability. Circular dichroism (CD) studies have demonstrated that melittin in an aqueous solution undergoes a transition from a helical tetramer to a random coil monomer not only by heating but also by cooling from room temperature (i.e., heat- and cold-denaturation, respectively). The heat-denaturation has been also examined by nuclear magnetic resonance (NMR) experiments, however, no NMR data have been presented on the cold-denaturation. In this paper, using proton (H) NMR spectroscopy, we show that melittin undergoes conformational transitions from the monomer to the tetramer to the monomer by elevating temperature from 2 to 70 °C. Only melittin including a trans proline peptide bond participates in the transitions, whereas melittin including a cis proline one does not. The tetramer has maximum conformation stability at around 20 °C, and cooperativity of the heat-denaturation is extremely low. [ABSTRACT FROM AUTHOR]

The M2 protein from influenza A is a proton channel as a tetramer, with a single transmembrane helix from each monomer lining the pore. Val27 and Trp41 form gates at either end of the pore and His37 mediates the shuttling of protons across a central barrier between the N-terminal and C-terminal aqueous pore regions. Numerous structures of this transmembrane domain and of a longer construct that includes an amphipathic helix are now in the Protein Data Bank. Many structural differences are apparent from samples obtained in a variety of membrane mimetic environments. High-resolution structural results in lipid bilayers have provided novel insights into the functional mechanism of the unique HxxxW cluster in the M2 proton channel. [Copyright &y& Elsevier]

Parkinson's disease is the second most common neurodegenerative disorder. Growing evidence indicates a causative role of misfolded forms of the protein ?-synuclein in the pathogenesis of Parkinson's disease. Intraneuronal aggregates of ?-synuclein occur in Lewy bodies and Lewy neurites, the cytopathological hallmarks of Parkinson's disease and related disorders called synucleinopathies. ?-Synuclein has long been defined as a 'natively unfolded' monomer of about 14?kDa (ref. 6) that is believed to acquire ?-helical secondary structure only upon binding to lipid vesicles. This concept derives from the widespread use of recombinant bacterial expression protocols for in vitro studies, and of overexpression, sample heating and/or denaturing gels for cell culture and tissue studies. In contrast, we report that endogenous ?-synuclein isolated and analysed under non-denaturing conditions from neuronal and non-neuronal cell lines, brain tissue and living human cells occurs in large part as a folded tetramer of about 58?kDa. Several methods, including analytical ultracentrifugation, scanning transmission electron microscopy and in vitro cell crosslinking confirmed the occurrence of the tetramer. Native, cell-derived ?-synuclein showed ?-helical structure without lipid addition and had much greater lipid-binding capacity than the recombinant ?-synuclein studied heretofore. Whereas recombinantly expressed monomers readily aggregated into amyloid-like fibrils in vitro, native human tetramers underwent little or no amyloid-like aggregation. On the basis of these findings, we propose that destabilization of the helically folded tetramer precedes ?-synuclein misfolding and aggregation in Parkinson's disease and other human synucleinopathies, and that small molecules that stabilize the physiological tetramer could reduce ?-synuclein pathogenicity. [ABSTRACT FROM AUTHOR]

Abstract: The structure, energetics, and vibrational spectra of the (HXeCN)2 dimer were investigated at the CCSD(T), MP2 and B3LYP levels. Such properties of the (HXeCN)3 trimer and (HXeCN)4 tetramer were investigated at the B3LYP level. The dimer, trimer, and tetramer were predicted to have a C 2h , C 2v , and D 2d structure, respectively. In all of these oligomers, the N⋯Xe intermonomeric interaction is the most important one for holding the monomers together. Included with the ZPVE and BSSE, the stabilization energy of the dimer is 12.36kcal/mol at the CCSD(T) level, while those of the dimer, trimer, and tetramer are 10.42, 18.23, and 31.34kcal/mol, respectively, at the B3LYP level. At the B3LYP level, with respect to those of the isolated monomer, the C−Xe and Xe−H asymmetric stretching frequencies are shifted by −11.2 and +128.0cm−1 for the dimer, −51.6, +220.7 and −11.5, +96.6cm−1 for the trimer, and −14.1 and +201.8cm−1 for the tetramer. [Copyright &y& Elsevier]

Abstract: Spiroplasmas belong to the class Mollicutes, representing the minimal, free-living, and self-replicating forms of life. Spiroplasmas are helical wall-less bacteria and the only ones known to swim by means of a linear motor (rather than the near-universal rotary bacterial motor). The linear motor follows the shortest path along the cell''s helical membranal tube. The motor is composed of a flat monolayered ribbon of seven parallel fibrils and is believed to function in controlling cell helicity and motility through dynamic, coordinated, differential length changes in the fibrils. The latter cause local perturbations of helical symmetry, which are essential for net directional displacement in environments with a low Reynolds number. The underlying fibrils'' core building block is a circular tetramer of the 59-kDa protein Fib. The fibrils'' differential length changes are believed to be driven by molecular switching of Fib, leading consequently to axial ratio and length changes in tetrameric rings. Using cryo electron microscopy, diffractometry, single-particle analysis of isolated ribbons, and sequence analyses of Fib, we determined the overall molecular organization of the Fib monomer, tetramer, fibril, and linear motor of Spiroplasma melliferum BC3 that underlies cell geometry and motility. Fib appears to be a bidomained molecule, of which the N-terminal half is apparently a globular phosphorylase. By a combination of reversible rotation and diagonal shift of Fib monomers, the tetramer adopts either a cross-like nonhanded conformation or a ring-like handed conformation. The sense of Fib rotation may determine the handedness of the linear motor and, eventually, of the cell. A further change in the axial ratio of the ring-like tetramers controls fibril lengths and the consequent helical geometry. Analysis of tetramer quadrants from adjacent fibrils clearly demonstrates local differential fibril lengths. [Copyright &y& Elsevier]

Peptidoglycan deacetlyase (HP0310, HpPgdA) from the gram-negative pathogen Helicobacter pylori, has been indicated as the enzyme responsible for a peptidoglycan modification that counteracts the host immune response. HpPgdA has been cloned, purified and expressed in good yield in E. coli. It has been crystallized, its structure determined and activity tests in vitro performed. The enzyme, which belongs to the polysaccharide deacetylases protein family, is a homo-tetramer. The four polypeptide chains, each folded into a single domain characterized by a non-canonical TIM-barrel fold, are arranged around a four-fold symmetry axis. The active site, one per monomer, contains a heavy ion coordinated in a way similar to other deacetylases. However, the enzyme showed no in vitro activity on the typical polysaccharide substrates of peptidoglycan deacetylases. In striking contrast with the known peptidoglycan deacetylases, HpPgdA does not exhibit a solvent-accessible polysaccharide binding groove, suggesting that the enzyme binds a small molecule at the active site. [ABSTRACT FROM AUTHOR]

Summary: Pyruvate carboxylase (PC) is a conserved multifunctional enzyme linked to important metabolic diseases. PC homotetramer is arranged in two layers with two opposing monomers per layer. Cryo-EM explores the conformational variability of PC in the presence of different substrates. The results demonstrate that the biotin-carboxyl carrier protein (BCCP) domain localizes near the biotin carboxylase (BC) domain of its own monomer and travels to the carboxyltransferase (CT) domain of the opposite monomer. All density maps show noticeable conformational differences between layers, mainly for the BCCP and BC domains. This asymmetry may be indicative of a coordination mechanism where monomers from different layers catalyze the BC and CT reactions consecutively. A conformational change of the PC tetramerization (PT) domain suggests a new functional role in communication. A long-range communication pathway between subunits in different layers, via interacting PT-PT and BC-BC domains, may be responsible for the cooperativity of PC from Staphylococcus aureus. [ABSTRACT FROM AUTHOR]

β-Amyloid (Aβ) is the main protein component of the amyloid plaques associated with Alzheimer's disease. Transthyretin (TTR) is a homotetramer that circulates in both blood and cerebrospinal fluid. Wild-type (wt) TTR amyloid deposits are linked to senile systemic amyloidosis. a common disease of aging, while several TTR mutants are linked to familial amyloid polyneuropathy. Several recent studies provide support for the hypothesis that these two amyloidogenic proteins interact, and that this interaction is biologically relevant. For example, upregulation of TTR expression in Tg2576 mice was linked to protection from the toxic effects of Aβ deposition [Stein, T. D., and Johnson, J. A. (2002) J. Neurosci. 22, 7380-7388]. We examined the interaction of Aβ with wt TTR as well as two mutants: F87M/LI10M. engineered to be a stable monomer, and TI 19M. a naturally occurring mutant with a tetrameric stability higher than that of the wild type. On the basis of enzyme-linked immunoassays as well as cross-linking experiments, we conclude that Aβ monomers bind more to TTR monomers than to TTR tetramers. The data further suggest that TTR tetramers interact preferably with Aβ aggregates rather than Aβ monomers. Through tandem mass spectrometry analysis of cross-linked TTR-Aβ fragments, we identified the A strand, in the inner β-sheet of TTR, as well as the EF helix, as regions of TTR that are involved with Aβ association. Light scattering and electron microscopy studies demonstrate that the outcome of the TTR-Aβ interaction strongly depends on TTR quaternary structure. While TTR tetramers may modestly enhance aggregation. TTR monomers decidedly arrest Aβ aggregate growth. These data provide important new insights into the nature of TTR- Aβ interactions. Such interactions may regulate TTR-mediated protection against Aβ toxicity. [ABSTRACT FROM AUTHOR]

Abstract: Copper complexes, [Cu(dm4bt)Cl(Hipht)] (1) and [{Cu(dm4bt)(H2O)(ipht)}4·2H2O] (2) (where dm4bt is 2,2′-dimethyl-4,4′-bithiazole, Hipht is hydrogen isophthalate and ipht is isophthalate) have been synthesized. These two complexes were characterized by IR, UV–Vis and EPR spectroscopy. Moreover; their single-crystal structures were studied by the X-ray diffraction method. Complex 1 has a monomer structure and copper has accepted a distorted square pyramidal structure. Isophthalic acid in 1 lost one of its protons and produced one bidentate carboxylate and one free carboxylic acid. Controlled deprotonation in the presence of ethylene diamine results in self-assemblies of 1 to form a tetramer complex of 2. Complex 2 has two kinds of spatial isomers which are resolved by EPR and X-ray. [Copyright &y& Elsevier]

Multinuclear NMR spectroscopic studies at low temperature (-110 to -150 °C) revealed that lithium p-fluorophenolate and the lithium enolates of cyclohexanone, cyclopentanone and 4-fluoroacetophe- none have tetrameric structures in THF/Et2O and THF/Et2O-HMPA by study of the effects of the addition of HMPA. The Z and E isomers of the lithium enolate of 1,3-bis-(4-fluorophenyl)-2-propanone (5F-Li) show divergent behavior. The Z isomer is completely dimeric in pure diethyl ether, and mostly dimeric in 3:2 THF/ether, where monomer could be detected in small amounts. TMTAN and PMDTA convert Z-5F-Li to a monomeric amine complex, and HMPA converts it partially to monomers, and partially to lithiate species (RO)2Li- and (RO)3Li2-. Better characterized solutions of these lithiates were prepared by addition of phosphazenium enolates (using P4-tBu base) to the lithium enolate in 1:1 ratio to form triple ion (RO)2Li- P4H+, or 2:1 ratio to form the higher lithiate (RO)3Li2- (P4H+)2) (quadruple ions). The E isomer of 5F-Li is also dimeric in 3:2 THF/Et2O solution, but is not detectably converted to monomer either by PMDTA or HMPA. In contrast to Z-5F-Li, the E isomer is tetrameric in diethyl ether even in the presence of excess HMPA. Thus for the two isomers of 5F six different enolate structures were characterized: tetramer, dimer, CIP-monomer, SIP-monomer, triple ion, and quadruple ion. [ABSTRACT FROM AUTHOR]

Potassium channels are tetrameric proteins that mediate K[sup+]-selective transmembrane diffusion. For KcsA, tetramer stability depends on interactions between permeant ions and the channel pore. We have examined the role of pore blockers on the tetramer stability of KirBac1.1. In 150 mM KCl, purified Kir- Bac1.1 protein migrates as a monomer (∼40 kDa) on SDSPAGE. Addition of Ba[sup2+] (K[sub1/2] ∼ 50 μM) prior to loading results in an additional tetramer band (∼160 kDa). Mutation A109C, at a residue located near the expected Ba[sup2+]-binding site, decreased tetramer stabilization by Ba[sup2+] (K[sub1/2] ∼ 300 μM), whereas I131C, located nearby, stabilized tetramers in the absence of Ba[sup2+]. Neither mutation affected Ba[sup2+] block of channel activity (using [sup86]Rb[sup+] flux assay). In contrast to Ba[sup2+], Mg[sup2+] had no effect on tetramer stability (even though Mg[sup2+] was a potent blocker). Many studies have shown Cd[sup2+] block of [sup2+] channels as a result of cysteine substitution of cavity-lining M2 (S6) residues, with the implicit interpretation that coordination of a single ion by cysteine side chains along the central axis effectively blocks the pore. We examined blocking and tetramer-stabilizing effects of Cd[sup2+] on KirBac1.1 with cysteine substitutions in M2. Cd[sup2+] block potency followed an α-helical pattern consistent with the crystal structure. Significantly, Cd[sup2+] strongly stabilized tetramers of I138C, located in the center of the inner cavity. This stabilization was additive with the effect of Ba[sup2+], consistent with both ions simultaneously occupying the channel: Ba[sup2+] at the selectivity filter entrance and Cd[sup2+] coordinated by I138C side chains in the inner cavity. [ABSTRACT FROM AUTHOR]

Abstract: Acidification of the transthyretin (TTR) tetramer facilitates dissociation and conformational changes in the protein, allowing alternatively folded monomers to self-assemble into insoluble amyloid fibers by a downhill polymerization mechanism in vitro. To investigate the influence of acidification on the quaternary and tertiary structures of TTR, crystal structures of wild-type human TTR at pH 4.0 and pH 3.5 have been determined to 1.7 Å resolution. The acidic pH crystals are isomorphous to most of the previously reported TTR structures, containing two subunits in the asymmetric unit (the so-called A and B subunits) but forming a tetramer through crystallographic symmetry. The pH 4.0 crystal structure reveals that the native fold of the tetramer remains mostly undisturbed. In particular, subunit A of the TTR pH 4.0 structure is very similar to the wild-type TTR pH 7.4 structure with an r.m.s.d. of 0.38 Å. In contrast, subunit B of the TTR pH 4.0 structure exhibits several significant changes. The EF-helix (residues 75–81) and the adjacent EF-loop (residues 82–90) show an r.m.s.d. greater than 2.0 Å. The acidic residues within this region (Glu72, Asp74, Glu89, and Glu92) undergo significant conformational changes that instigate movement of the EF helix–loop region and make residues Lys70, Lys76, His88, and His90 orient their side chains toward these acidic residues. In particular, Glu89 undergoes a maximum deviation of 5.6 Å, occupying Phe87''s initial position in the wild-type TTR pH 7.4 structure, and points its side chain into a hydrophobic pocket of the neighboring subunit. In the pH 3.5 structure, the EF helix–loop region is completely disordered. These results demonstrate that acidic conditions increase the susceptibility of the EF helix–loop region of the TTR B subunit to undergo conformational changes and unfold, likely destabilizing the tetramer and identifying at least the initial conformational changes likely occurring within the tetramer that leads to the amyloidogenic monomer. [Copyright &y& Elsevier]

l-Threonine dehydrogenase (TDH) is an enzyme that catalyzes the oxidation of l-threonine to 2-amino-3-ketobutyrate. We solved the first crystal structure of a medium chain l-threonine dehydrogenase from a hyperthermophilic archaeon, Pyrococcus horikoshii (PhTDH), by the single wavelength anomalous diffraction method using a selenomethionine-substituted enzyme. This recombinant PhTDH is a homo-tetramer in solution. Three monomers of PhTDHs were located in the crystallographic asymmetric unit, however, the crystal structure exhibits a homo-tetramer structure with crystallographic and non-crystallographic 222 symmetry in the cell. Despite the low level of sequence identity to a medium-chain NAD(H)-dependent alcohol dehydrogenase (ADH) and the different substrate specificity, the overall folds of the PhTDH monomer and tetramer are similar to those of the other ADH. Each subunit is composed of two domains: a nicotinamide cofactor (NAD(H))-binding domain and a catalytic domain. The NAD(H)-binding domain contains the α/β Rossmann fold motif, characteristic of the NAD(H)-binding protein. One molecule of PhTDH contains one zinc ion playing a structural role. This metal ion exhibits coordination with four cysteine ligands and some of the ligands are conserved throughout the structural zinc-containing ADHs and TDHs. However, the catalytic zinc ion that is coordinated at the bottom of the cleft in the case of ADH was not observed in the crystal of PhTDH. There is a significant difference in the orientation of the catalytic domain relative to the coenzyme-binding domain that results in a larger interdomain cleft. [Copyright &y& Elsevier]

The homotetrameric protein transthyretin (TTR) must undergo rate-limiting dissociation to its constituent monomers in order to enable partial denaturation that allows the process of amyloidogenesis associated with human pathology to ensue. The TTR quaternary structure contains two distinct dimer interfaces, one of which creates the two binding sites for the natural ligand thyroxine. Tetramer dissociation could proceed through three distinct pathways; scission into dimers along either of the two unique quaternary interfaces followed by dimer dissociation represents two possibilities. Alternatively, the tetramer could lose monomers sequentially. To elucidate the TTR dissociation pathway, we employed two different TTR constructs, each featuring covalent attachment of proximal subunits. We demonstrate that tethering the A and B subunits of TTR with a disulfide bond (as well as the symmetrically disposed C and D subunits) allows urea-mediated dissociation of the resulting (TTR-S-S-TTR)2 construct, affording (TTR-S-S-TTR)1 retaining a stable 16-stranded β-sheet structure that is equivalent to the dimer not possessing a thyroid binding site. In contrast, linking the A and C subunits employing a peptide tether (TTR-L-TTR)2 affords a kinetically stable quaternary structure that does not dissociate or denature in urea. Both tethered constructs and wild-type TTR exhibit analogous stability based on guanidine hydrochloride denaturation curves. The latter denaturant can denature the tetramer, unlike urea, which can only denature monomeric TTR; hence urea requires dissociation to monomers to function. Under native conditions, the (TTR-S-S-TTR)2 construct is able to dissociate and incorporate subunits from labeled WT TTR homotetramers at a rate equivalent to that exhibited by WT TTR. In contrast, the (TTR-L-TTR)2 construct is unable to exchange any subunits, even after 180 h. All of the data presented herein and elsewhere demonstrate that the pathway of TTR tetramer dissociation occurs by scission of the tetramer along the crystallographic C2 axis affording AB and CD dimers that rapidly dissociate into monomers. Determination of the mechanism of dissociation provides an explanation for why small molecules that bind at the AB/CD dimer-dimer interface impose kinetic stabilization upon TTR and disease-associated variants thereof. [ABSTRACT FROM AUTHOR]

The structure of human translin at 2.2 Å resolution is reported in space group C2221. Translin forms a tetramer in the asymmetric unit. Although the monomer structure is almost the same as the crystal structure of murine translin in space group P21212, the relative positions of the tetramers differ between the human and murine translins. This suggests that the multimerization of translin is flexible; the flexibility may be related to the binding to DNA/RNA. [ABSTRACT FROM AUTHOR]

The iron- and manganese-containing superoxide dismutases (Fe/Mn-SOD) share the same chemical function and spatial structure but can be distinguished according to their modes of oligomerization and their metal ion specificity. They appear as homodimers or homotetramers and usually require a specific metal for activity. On the basis of 261 aligned SOD sequences and 12 superimposed x-ray structures, two phenetic trees were constructed, one sequence-based and the other structurebased. Their comparison reveals the imperfect correlation of sequence and structural changes; hyperthermophilicity requires the largest sequence alterations, whereas dimer/tetramer and manganese/iron specificities are induced by the most sizable structural differences within the monomers. A systematic investigation of sequence and structure characteristics conserved in all aligned SOD sequences or in subsets sharing common oligomeric and/or metal specificities was performed. Several residues were identified as guaranteeing the common function and dimeric conformation, others as determining the tetramer formation, and yet others as potentially responsible for metal specificity. Some form cation-π interactions between an aromatic ring and a fully or partially positively charged group, suggesting that these interactions play a significant role in the structure and function of SOD enzymes. Dimer/tetramer- and iron]manganese-specific fingerprints were derived from the set of conserved residues; they can be used to propose selected residue substitutions in view of the experimental validation of our in silico derived hypotheses. [ABSTRACT FROM AUTHOR]

The misfolding of transthyretin (TTR), including rate-limiting tetramer dissociation and partial monomer denaturation, is sufficient for TTR misassembly into amyloid and other abnormal quaternary structures associated with senile systemic amyloidosis, familial amyloid polyneuropathy, and familial amyloid cardiomyopathy. Monovalent small molecules that bind to one or both of the unoccupied thyroid hormone binding sites at the TTR quaternary structure interface stabilize the native state, raising the kinetic barrier for tetramer dissociation sufficiently that the rate of dissociation, and therefore amyloidosis, becomes slow. Bivalent amyloid inhibitors that bind to both binding sites simultaneously are reported herein. The candidate bivalent inhibitors are generally unable to bind to the native TTR tetramer and typically do not engage in monovalent binding owing to a strong inhibitor orientation preference. However, the TTR quaternary structure can assemble around several of the bivalent inhibitors if the inhibitor intercepts the protein before assembly occurs. Some of the wild-type TTR.bivalent inhibitor complexes prepared in this fashion retain a tetrameric structure when subjected to substantial denaturation stresses (8 M urea, 120 h). The best bivalent inhibitor reduced acid-mediated TTR (3.6 μM) amyloid fibril formation to 6% of that exhibited by TTR in the absence of inhibitor, a significant improvement over the 30% observed for the best monovalent inhibitors (3.6 μM, 72 h). The apparent dissociation rate of the best bivalent inhibitor is effectively zero, consistent with the idea that TTR tetramer dissociation and inhibitor dissociation are linked--as a result of the inhibitor-templating tetramer assembly. X-ray cocrystal structures of two of the complexes demonstrate that the bivalent inhibitors simultaneously occupy both sites in TTR, consistent with the 1:1 binding stoichiometry derived from HPLC analysis. The purpose of this study was to demonstrate that bivalent inhibitors could be useful; what resulted are the best inhibitors produced to date. In this context, molecules capable of intercepting TTR during folding and assembly in the lumen of the endoplasmic reticulum would be of obvious interest. [ABSTRACT FROM AUTHOR]

A variety of human haemoglobins (Hbs) are produced at different stages of human development, including three embryonic Hbs, foetal Hb and adult Hb. All are heterotetramers. During crystallization experiments on human embryonic Hb Portland (ζ2γ2), it was discovered by crystallographic and biochemical analysis that the homotetramer Hb Bart's (γ4) preferentially crystallizes from ζ2γ2 solutions below pH 5. This results from dissociation of Hb Portland into γ2 dimers and ζ monomers and has interesting implications for subunit interactions and tetramer stability in Hbs. It also makes possible a full crystallographic analysis of Hb Bart's, which is of considerable medical significance because of its presence in the red blood cells of millions of people worldwide who suffer from α-thalassaemia. [ABSTRACT FROM AUTHOR]

Discusses the effects of mutations in apyrase conserved region determined by whether they occur in intact tetrameres or in disrupted tetrameres or monomers. Result of substitution in tetrameres lacking either transmembrane domain and in monomers lacking transmembrane domains; Varied adenosine triphosphatase and adenosine diphosphatase mechanisms used by CD39.

Describes the identification of the tetramer within the crystal packing and structure of the monomers and their interactions in the tetramer. Identification of the relevant tryptase tetramer; Description of the tetramer structure; Structure of the monomer; Interaction with substrates and inhibitors.

Aquaporins form a large family of membrane channels involved in osmoregulation. Electron crystallography has shown monomers to consist of six membrane spanning α-helices confirming sequence based predictions. tions. Surface exposed loops are the least conserved regions, allowing differentiation of aquaporins. Atomic force microscopy was used to image the surface of aquaporin Z, the water channel of Escherichia coil Recombinant protein with an N-terminal fragment including 10 histidines was isolated as a tetramer by Ni-affinity chromatography, and reconstituted into two- Dimensional crystals with p4212 symmetry. Small crystalline areas with p4 symmetry were found as well. Imaging both crystal types before and after cleavage of the N-termini allowed the cytoplasmic surface to be identified; a drastic change of the cytoplasmic surface accompanied proteolytic cleavage, while the extracellular surface morphology did not change. Flexibility mapping and volume calculations identified the longest loop at the extracellular surface. This loop exhibited a reversible force-induced conformational change. [ABSTRACT FROM AUTHOR]

The p53 tumor suppressor protein is a tetramer that binds sequence-specifically to a DNA consensus sequence consisting of two consecutive half-sites, with each half-site being formed by two head-to-head quarter-sites (→← →←). Each p53 subunit binds to one quarter-site, resulting in all four DNA quartersites being occupied by one p53 tetramer. The tetramerization domain forms a symmetric dimer of dimers, and two contrasting models have the two DNA-binding domains of each dimer bound to either consecutive or alternating quarter-sites. We show here that the two monomers within a dimer bind to a half-site (two consecutive quarter-sites), but not to separated (alternating) quarter-sites. Tetramers bind similarly, with the two dimers within each tetramer binding to pairs of half-sites. Although one dimer within the tetramer is sufficient for binding to one half-site in DNA, concurrent interaction of the second dimer with a second half-site in DNA drastically enhances binding affinity (at least 50-fold). This cooperative dimer-dimer interaction occurs independently of tetramerization and is a primary mechanism responsible for the stabilization of p53 DNA binding. Based on these findings, we present a model of p53 binding to the consensus sequence, with the tetramer binding DNA as a pair of clamps. [ABSTRACT FROM AUTHOR]

The hydrolysis of asparagine and glutamine by L-asparaginase has been used to treat acute lymphoblastic leukemia for over four decades. Each L-asparaginase monomer has a long loop that closes over the active site upon substrate binding, acting as a lid. Here we present a comparative study of two commercially available preparations of the drug containing Escherichia coli L-Asparaginase 2 (EcA2), performed by a comprehensive array of biophysical and biochemical approaches. We report the oligomeric landscape and conformational and dynamic plasticity of E. coli type 2 L-asparaginase present in two different formulations, and its relationship with L-aspartic acid, which is present in Aginasa, but not in Leuginase. The L-Asp present in Aginasa formulation was found to provide to EcA2 a resistance to in vitro proteolysis. EcA2 shows a composition of monomers and oligomers up to tetramers, which is mostly not altered in the presence of L-Asp. Ion-mobility spectrometry–mass spectrometry reveals two conformers for the monomeric EcA2, and that monomeric species has sufficient capacity for selective binding to L-Asp and L-Glu. The N-terminal loop of the EcA2 present in Leuginase, which is part of the active site is disordered, but it gets ordered in the presence of L-Asp, while L-Glu only does so to a limited extent. These data provide new insights on the mechanistic of ligand recognition by EcA2, and the impact of formulation in its conformational diversity landscape. [Display omitted] • L-asparaginase 2 from E. coli (EcA2) is crucial in the treatment of acute lymphoblastic leukemia. • Two EcA2 products were found to be dissimilar in formulative composition and oligomeric distribution in solution. • Monomeric EcA2 was found to populate two conformers, and binding selectively to aminoacids, not requiring supramolecular assembly. • L-Asp reorganizes the active site 'lid', while L-Glu orders the 'lid' only partially, exerting crucial role in the susceptibility to limited proteolysis of EcA2 in vitro. • Despite equivalent sequence and high-resolution structure, formulative design determine the conformational and stability landscape. [ABSTRACT FROM AUTHOR]

Abstract: It has recently been reported that tropomyosin exists exclusively as a dimer in physiological salt conditions. It is shown in the present work using analytical ultracentrifugation that, on the contrary, tropomyosin is in equilibrium between monomer, dimer and tetramer with a weak tendency to dimerize and tetramerize. Such a finding has consequences for the assembly of the tropomyosin–actin complex. [Copyright &y& Elsevier]

p53 is a tetrameric multidomain protein that triggers the anticancer cellular response to stress. We have calculated a three-dimensional reconstruction of full-length human p53 in the absence of DNA using single-particle electron microscopy. The reconstruction of DNA-free full-length p53 shows a square-shaped structure with four distinct domains and a hollow center. In comparison with the known compacted DNA-bound full-length p53 structures, the DNA-free p53 tetramer adopts a relaxed conformation with separated monomers and oligomerization interfaces different from those of the DNA-bound conformation. [ABSTRACT FROM AUTHOR]

Manganese superoxide dismutase (MnSOD) from different species differs in its efficiency in removing high concentrations of superoxide (O2-), due to different levels of product inhibition. Human MnSOD exhibits a substantially higher level of product inhibition than the MnSODs from bactena. In order to investigate the mechanism of product inhibition and whether it is a feature common to eukaryotic MnSODs, we purified MnSOD from Saccharomyces cerevisiae (ScMnSOD). It was a tetramer with 0.6 equiv of Mn per monomer. The catalytic activity of ScMnSOD was investigated by pulse radiolysis and compared with human and two bacterial (Escherichia coli and Deinococcus radiodurans) MnSODs. To our surprise, ScMnSOD most efficiently facilitates removal of high concentrations of O2- among these MnSODs. The gating value k2/k3 that characterizes the level of product inhibition scales as ScMnSOD > D. radiodurans MnSOD > E. coil MnSOD > human MnSOD. While most MnSODs rest as the oxidized form, ScMnSOD was isolated in the Mn2+ oxidation state as revealed by its optical and electron paramagnetic resonance spectra. This finding poses the possibility of elucidating the origin of product inhibition by comparing human MnSOD with ScMnSOD. [ABSTRACT FROM AUTHOR]

Summary Biogenesis and dynamics of thylakoid membranes likely involves membrane fusion events. Membrane attachment of the inner membrane-associated protein of 30 kDa (IM30) affects the structure of the lipid bilayer, finally resulting in membrane fusion. Yet, how IM30 triggers membrane fusion is largely unclear. IM30 monomers pre-assemble into stable tetrameric building blocks, which further align to form oligomeric ring structures, and differently sized IM30 rings bind to membranes. Based on a 3D reconstruction of IM30 rings, we locate the IM30 loop 2 region at the bottom of the ring and show intact membrane binding but missing fusogenic activity of loop 2 mutants. However, helix 7, which has recently been shown to mediate membrane binding, was located at the oppossite, top side of IM30 rings. We propose that a two-sided IM30 ring complex connects two opposing membranes, finally resulting in membrane fusion. Thus, IM30-mediated membrane fusion requires a Janus-faced IM30 ring. [ABSTRACT FROM AUTHOR]

replying to J. Burré . Nature 498, http://dx.doi.org/10.1038/nature12125 (2013)In disagreeing with our report that native α-synuclein occurs physiologically as an α-helically folded tetramer in neural and erythroid cells, Burré et al. conclude instead that 'native brain α-synuclein' consists of a largely unstructured monomer. They make two implications about our paper that are inaccurate: (1) that our findings pertained only to erythrocyte α-synuclein (we reported multiple experiments on neural cells); and (2) that we concluded that cellular α-synuclein is a stable tetramer under all conditions (we did not use the term 'stable', and we observed monomers and some other oligomers in normal cells (e.g., Fig. 1d of ref. 1)). Indeed, we emphasized the need to discover 'compounds that ... could kinetically stabilize native tetramers and prevent pathogenic α-synuclein aggregation'. Although the data in our report suggest that tetramers are the predominant native species, tetramers and other oligomers arise from monomers, so there must be an equilibrium between monomeric and oligomeric forms in cells. Pathogenic events (e.g., mutations) could alter this equilibrium, and some therapeutic compounds could potentially re-establish it, as we explicitly suggested. [ABSTRACT FROM AUTHOR]

Current dogma for transthyretin (TTR) pathogenesis is that mutations in TTR alter its structure such that the tetramer becomes unstable and prone to release of monomer which then becomes the putative building block of the fibril. This hypothesis is supported by thermodynamic data showing decreased stability of mutant TTR tetrameric proteins and accelerated fibril formation under acidic conditions in vitro. There are, however, a number of questions that are not readily answered by this simplistic model of a very complex disease. Worrisome questions still to be answered include: 1. If the monomer is the precursor of the fibril, why do fibril deposits contain large amounts of wild-type TTR and not just variant? 2. If destabilized tetramers can form fibrils in vitro, why do we consistently find partial proteolysis of fibril subunit proteins? If enzymatic proteolysis is a required step in fibril formation, are the findings of in vitro fibril formation relevant to the true pathogenesis? 3. With some TTR mutations (e.g. 122?Val), it would appear that very little TTR is present in the blood (probably due to degradation prior to hepatic secretion). Enough mutant TTR circulates to the heart and nerves to cause pathology but, if the mutant only serves to initiate fibril deposition, why are not the deposits mainly wild-type TTR? 4. Since mutated TTR is present from birth, why is TTR amyloidosis of such delayed onset? What is the role of aging factors? 5. Do the variations in biochemical analyses of heart and nerve versus choroid and leptomeningeal fibrils tell us something about pathogenesis? These are questions we need to address. Do not expect quick and easy answers. Hopefully, they will generate thought and discussion. [ABSTRACT FROM AUTHOR]

Transthyretin (TTR) is a tetrameric beta-sheet-rich protein. Its deposits have been implicated in four different amyloid diseases. Although aggregation of the wild-type sequence is responsible for the senile form of the disease, more than one hundred variants have been described thus far, most of which confer a more amyloidogenic character to TTR, mainly because they compromise the stability of the protein in relation to monomer formation, which upon misfolding is intrinsically aggregation-prone. We report the case of a Brazilian patient suffering from a severe cardiomyopathy who carries a rare mutation in exon 2 of the TTR gene that results in an Ala to Asp substitution at position 19 (A19D). The putative pathogenic mechanisms of this variant were analyzed in silico. We constructed a structural model for the A19D tetramer from which its thermodynamic stability was compared to that displayed by the V30M (more amyloidogenic than WT-TTR) and T119M (non-amyloidogenic) variants. The FoldX force field predicted that A19D and V30M are 10.88 and 8.07 kCal/mol less stable than the WT-TTR, while T119M is 5.15 kCal/mol more stable, which is consistent with the aggregation propensities exhibited by these variants. We analyzed the step in which the tetramer-dimer-monomer-unfolded monomer equilibrium might contribute the most to the increased or decreased amyloidogenicity in each variant. Our results suggest that the concentration of four non-native negative charges occur inside thyroxine-binding channels, and the loss of contacts at both the tetrameric and dimeric interfaces would account for an overall decreased stability of the tetramer and the consequent enhanced amyloidogenicity of the A19D variant. As far as we know, this is the first description of a non-V30M mutation in Brazil. [ABSTRACT FROM AUTHOR]

Hemoglobin dissociation is of great interest in protein process and clinical medicine as well as in artificial blood research. However, the pathway and mechanisms of pH-dependent human Hb dissociation are not clear, whether Hb would really dissociate into monomers is still a question. Therefore, we have conducted a multi-technique investigation on the structure and function of human Hb versus pH. Here we demonstrate that tetramer hemoglobin can easily dissociate into dimer in abnormal pH and the tetramer → dimer dissociation is reversible if pH returns to normal physiological value. When the environmental pH becomes more acidic (<6.5) or alkaline (>8.0), Hb can further dissociate from dimer to monomer. The proportion of monomers increases while the fraction of dimers decreases as pH declines from 6.2 to 5.4. The dimer → monomer dissociation is accompanied with series changes of protein structure thus it is an irreversible process. The structural changes in the dissociated Hbs result in some loss of their functions. Both the Hb dimer and monomer cannot adequately carry and release oxygen to the tissues in circulation. These findings provide a comprehensive understanding on the pH-dependent protein transitions of human Hb, give guideline to explain complex protein processes and the means to control protein dissociation or re-association reaction. They are also of practical value in clinical medicine, blood preservation and blood substitute development. [ABSTRACT FROM AUTHOR]