Induced circular dichroism and mode of binding of acridine orange adsorbed on β-form poly(S-carboxyethyl-L-cysteine) in aqueous solutions

The absorption spectra and circular dichroism (CD) have been measured for aqueous solutions of acridine orange of a constant concentration, [D] = 5 × 10^?5M, mixed with poly(S-carboxyethyl-L -cysteine) in various mixing ratios, [P]/[D], ranging from 330 to 11, at different pH. The absorption spectra of the dye–polymer solutions are hypochromic, and the main band is located at 470 nm, accompanying a shoulder at 500 nm. At alkaline pH, no CD is induced in the visible region. At neutral and acidic pH, where the polymer is in the β-conformation, CD is induced in the visible and near-uv regions. A pair of CD bands is located at the region around 450 nm, when the pH is around the neutrality, while it appears at the region around 500 nm at acidic pH. Thus, the optically active species of bound dye changes from dimer to monomer on lowering the pH. These species form dissymmetric arrays along a polypeptide chain. The fraction of bound dye forming dissymmetric sequences is not high, but most of bound dye is adsorbed randomly on the ionized carboxyl groups of polypeptide chain and gives rise to hypochromism only. A dissymmetric structure of dye–polymer complexes is presented, in which the polymer has the β-conformation and the dye cations, either dimeric or monomeric, bind to its side chains, in such a way that the longer axes of molecular planes of bound dye form a two-fold, right-handed helix along the extended polypeptide chain. A zeroth-order calculation of CD based on the coupled oscillator model leads to the result that each dissymmetric array of dye consists, on the average, of two dimeric or monomeric cations. This low number of bound cations in a dissymmetric array and the large fraction of randomly adsorbed dye suggest that the hydrophobic interaction of dye with the polymer is strong, so that dye cations are adsorbed sparsely on both sides of the extended polypeptide chain.     

An examination of structural interactions presumed to be of importance in the stabilization of phospholipase A2 dimers based upon comparative protein sequence analysis of a monomeric and dimeric enzyme from the venom ofAgkistrodon p. piscivorus

Phospholipases A₂ may exist in solution both as monomers and dimers, but enzymes that form strong dimers (K _D approximately 10^–9 M) have been found, thus far, only in venoms of the snake family Crotilidae. The complete amino acid sequences of a basic monomeric and an acidic dimeric phospholipase A₂ fromAgkistrodon piscivorus piscivorus (American cotton-mouth water moccasin) venom have been determined by protein sequencing methods as part of a search for aspects of structure contributing to formation of stable dimers. Both the monomeric and dimeric phospholipases A₂ are highly homologous to the dimeric phospholipases A₂ fromCrotalus atrox andCrotalus adamanteus venoms, and both have the seven residue carboxy-terminal extension characteristic of the crotalid and viperid enzymes. Thus, it is clear that the extension is not a prerequisite for dimerization. Studies to date have revealed two characteristic features of phosphilipases A₂ that exist in solution as strong dimers. One is the presence in the dimers of a Pro-Pro sequence at position 112 and 113 which just precedes the seven residue carboxy-terminal extension (residues 116–122). The other is a low isoelectric point; only the acidic phospholipases A₂ have been observed, thus far, to form stable dimers. These, alone or together, may be necessary, though not sufficient conditions for phospholipase A₂ dimer formation. Ideas regarding subunit interactions based upon crystallographic data are evaluated relative to the new sequence information on the monomeric and dimeric phospholipases A₂ fromA. p. piscivorus venom.     

Effects of various forms of surfactant protein C on tidal volume in ventilated immature newborn rabbits.

Surfactant protein (SP)-C is characterized by alpha-helix structure and palmitoyl groups attached to two cysteine residues. We examined the function of palmitoylation and dimerization in promotion of tidal volume in immature newborn rabbits. Reconstituted surfactants were made from a mixture of synthetic phospholipids and porcine SP-B (basic mixture) by adding various forms of SP-Cs: normal SP-C isolated from porcine lungs and monomeric or dimeric forms of SP-C. These latter two were isolated from patients with pulmonary alveolar proteinosis and were less palmitoylated. Animals were ventilated at an inspiratory pressure of 25 cmH2O. Median tidal volumes were <2 ml/kg in nontreated controls, 7.7 ml/kg in animals receiving the basic mixture without SP-C, and >18 ml/kg in animals treated with reconstituted surfactants containing 3% normal or 2% dimeric SP-C (P < 0.05 vs. basic mixture). The physiological effect of basic mixture was not improved by monomeric SP-C. We conclude that palmitoyl groups are important for the physiological effects of SP-C and that the dimeric form also improves physiological effects.     

Semisynthesis and properties of some insulin analogs

The trypsin-catalyzed coupling of bovine (Boc)₂-desoctapeptide (B23-B30)-insulin with synthetic octapeptides, H-Gly-X₂-X₃-X₄-Thr-Pro-Lys(Boc)-Thr-OH (X₂ = Phe or Ala, X₃ = Phe or Ala, X₄ = Tyr or Ala), followed by deprotection and purification produced the [Ala^B24, Thr^B30]-, [Ala^B25, Thr^B30]-, and [Ala^B26, Thr^B30]-analogs of bovine insulin in yields of 32, 35, and 32%, respectively. The biological activity of these analogs decreased in the order, normal insulin ([Thr^B30]-bovine insulin) = Ala^B26-insulin > Ala^B25-insulin > Ala^B24-insulin, as assayed for receptor binding and some other biological effects, in contrast with the corresponding Leu-analogs of human insulin, in which the activity decreased in the order, normal insulin > Leu^B24-insulin > Leu^B25-insulin. The affinity to insulin antibodies greatly diminished in both Ala^B24-insulin and Leu^B24-insulin but not in the B25-substituted analogs. The CD spectra of the Leu- and the Ala-analogs were compared with those of normal insulins to show that no apparent correlation seems to exist between the decrease in biological activity and the conformational changes observed in solution. The effects of organic solvents on the peptide-bond equilibrium and on the stability of trypsin are also discussed.     

Synthetic Analogues of Conantokin-G: NMDA Antagonists Acting Through a Novel Polyamine-Coupled Site

Abstract: Conantokin-G (con-G) is a 17-amino-acid polypeptide that acts as an N-methyl-d -aspartate (NMDA) antagonist. This action has been attributed to a specific but noncompetitive inhibition of the positive modulatory effects of polyamines at NMDA receptors. Con-G possesses several unusual structural features, including five γ-carboxyglutamate (Gla) residues and a high degree of helicity in aqueous media. Previous structure-activity studies indicated that one or more Gla residues are necessary for NMDA antagonist activity. Con-G analogues were synthesized with alanine (Ala), serine (Ser), and phosphoserine substituted for Gla to assess the contribution of individual Gla residues to biological activity and secondary structure. Replacement of Gla in positions 3 and 4 resulted in polypeptides with markedly reduced and no NMDA antagonist actions, respectively. In contrast, Gla residues in positions 7, 10, and 14 are not required for NMDA antagonist actions because the potencies of con-G analogues containing Ser⁷, Ser¹⁰, Ala¹⁴, and Ser¹⁴ to inhibit spermine-stimulated [³H]MK-801 binding are similar to the parent peptide. Moreover, the Ala⁷ derivative of con-G was about fourfold more potent than the parent peptide both as an inhibitor of spermine-stimulated increases in [³H]MK-801 binding (IC₅₀ of ～45 nM) and in reducing NMDA-stimulated increases in cyclic GMP levels (IC₅₀ of ～77 nM) in cerebellar granule cell cultures. Although con-G and its analogues assumed mixtures of 3₁₀ and α-helices, no clear-cut relationship was evinced between the NMDA antagonist properties of these peptides and the degree of helicity they assumed in aqueous solutions. Together with the inability of con-G to affect 5,7-dichloro[³H]kynurenic acid, [³H]CGP-39653, and [³H]ifenprodil binding, these data are consistent with the hypothesis that this polypeptide acts at a unique, polyamine-associated site on NMDA receptors.     

Design and synthesis of dipeptide mimetics of the brain-derived neurotrophic factor

Low-molecular-weight mimetics of loops 1 and 4 of the brain-derived neurotrophic factor (BDNF) have been designed and synthesized. The compounds represent monomeric and dimeric amides of N-acyldipeptides. Their dipeptide fragments coincide in sequence with the central regions of beta-turns of the corresponding neurotrophin loops, and acyl groups are the bioesosteres of preceding amino acid residues. Hexa- or heptamethylenediamines were used as spacers to link the C-terminal regions of dipeptides in dimeric mimetics of BDNF. These compounds were synthesized by classical methods of peptide synthesis in solution and received the laboratory codes GSB-104 (HO-Suc-Ser-Lys-NH₂), GSB-106 {[HO-Suc-Ser-Lys-NH-(CH₂)₃?]₂}, GSB-207 (HO-Suc-Met-Ser-NH₂), and GSB-214 ([HO-Suc-Met-Ser-NH-(CH₂)_7/2-]₂). It was shown using immortalized hippocampal cells of the HT22 line under conditions of oxidative stress that the dimeric mimetics of both loops at concentrations of 10^?5?10^?8 M possess a neuroprotective activity. The monomeric loop 1 mimetic GSB-207 in the same concentration range is inactive, and the monomeric loop 4 mimetic GSB-104 at a concentration of 10^?7 impairs the survival of neurons. The finding that only dimeric mimetics possess the neuroprotective activity is consistent with the data indicating that BDNF is active in the homodimeric form. As opposed to the dimeric loop 1 mimetic GSB-214, the dimeric loop 4 mimetic GSB-106 exhibits the antidepressant activity typical for BDNF in the Porsolt test on rats at doses of 0.1 and 1 mg/kg injected intraperitoneally. This suggests that the antidepressant activity of BDNF is related to its 4th loop. We believe that the compounds obtained will be useful in studies of the mechanism of action of BDNF and may form the basis for the design of a novel group of drugs with antidepressant and neuroprotective activities.     

Biophysical studies of the membrane-embedded and cytoplasmic forms of the glucose-specific Enzyme II of the E. coli phosphotransferase system (PTS)

The glucose Enzyme II transporter complex of the Escherichia coli phosphotransferase system (PTS) exists in at least two physically distinct forms: a membrane-integrated dimeric form, and a cytoplasmic monomeric form, but little is known about the physical states of these enzyme forms. Six approaches were used to evaluate protein-protein and protein-lipid interactions in this system. Fluorescence energy transfer (FRET) using MBP-II(Glc)-YFP and MBP-II(Glc)-CFP revealed that the homodimeric Enzyme II complex in cell membranes is stable (FRET(-)) but can be dissociated and reassociated to the heterodimer only in the presence of Triton X100 (FRET(+)). The monomeric species could form a heterodimeric species (FRET(+)) by incubation and purification without detergent exposure. Formaldehyde cross linking studies, conducted both in vivo and in vitro, revealed that the dimeric MBP-II(Glc) activity decreased dramatically with increasing formaldehyde concentrations due to both aggregation and activity loss, but that the monomeric MBP-II(Glc) retained activity more effectively in response to the same formaldehyde treatments, and little or no aggregation was observed. Electron microscopy of MBP-II(Glc) indicated that the dimeric form is larger than the monomeric form. Dynamic light scattering confirmed this conclusion and provided quantitation. NMR analyses provided strong evidence that the dimeric form is present primarily in a lipid bilayer while the monomeric form is present as micelles. Finally, lipid analyses of the different fractions revealed that the three lipid species (PE, PG and CL) are present in all fractions, but the monomeric micellar structure contains a higher percentage of anionic lipids (PG & CL) while the dimeric bilayer form has a higher percentage of zwitterion lipids (PE). Additionally, evidence for a minor dimeric micellar species, possibly an intermediate between the monomeric micellar and the dimeric bilayer forms, is presented. These results provide convincing evidence for interconvertible physical forms of Enzyme-II(Glc).     

Observations on the formation of deletions on monomeric and dimeric plasmids in Escherichia coli

We have studied the formation of spontaneous mutations on plasmids present In the monomeric and dimeric states in a recF strain of Escherichia coli. Two test systems were employed: (i) the precise excision of Tn5 from the tetA gene of the plasmid pBR322 and (ii) operator constitutive (O^c) mutations on the pBR322-derived plasmid pPY97. The rate of O^c mutations was increased by a factor of three when this plasmid was present in the dimeric state compared to the monomeric state and the O^c phenotype was caused by small deletions in the operator sequence. No apparent mutational hot-spot was found. The rate of Tn5 excision was increased on dimeric compared to monomeric plasmids. Excision from a dimeric plasmid usually resulted in two types of mutant plasmids; a dimeric plasmid, where the Tn5 had excised from one of the plasmid units, and a monomeric parental pBR322. A mechanism to account for this is suggested. Complementation tests revealed that the increased mutation rate on dimeric plasmids is the result of dimers being mutaphilic per se, rather than the result of a general, trans-acting increase in mutation rates of the host, induced by the presence of the dimeric plasmid. Furthermore, it was found that the rate of Tn5 excision from plasmids in the monomeric state was increased when the region carrying the inserted Tn5 was duplicated.     

Chemical, physical, and biological characterization of a dimeric form of biosynthetic human growth hormone

A dimer of biosynthetic human growth hormone (HGH) has been isolated and characterized. This entity, which is the predominant dimeric species in biosynthetic HGH, is chemically identical to monomeric HGH and exists in a noncovalent dimeric form which is dissociated to monomeric HGH on polyacrylamide electrophoresis gels or in aqueous solutions containing 30% acetonitrile. This substance, found in all production lots of pituitary HGH, biosynthetic HGH, and biosynthetic methionyl HGH examined, is much less biopotent than monomeric HGH and can be distinguished from monomeric HGH by a monoclonal antibody. These data demonstrate that polyacrylamide gel electrophoresis is not a valid method for measuring this dimer and that size-exclusion chromatography under aqueous conditions is required.     

The hemoglobins of phoronopsis viridis, of the primitive invertebrate phylum phoronida: characterization and subunit structure.

The primitive invertebrate, Phoronopsis viridis, of the phylum Phoronida, has intra-cellular hemoglobins composed of four unique polypeptide chains, two of which associate to form hetero- and homodimers and two which do not associate at all. The CO-derivatives of the associating chains are completely dimeric; removal of the ligand does not result in further aggregation as it does in several other invertebrate hemoglobins. Oxidation of the associating hemoglobins is accompanied by dissociation to monomers, but the cyanide derivative of the methemoglobin is dimeric. The four polypeptide chains all have molecular weights of about 16,000 as determined by iron content and gel electrophoresis with sodium dodecyl sulfate. The two associating chains form three components with isoelectric points at pH 5.6, 5.9, and 6.9 whereas those for the two monomeric chains are at pH 6.2 and 7.9. The chains have been characterized by amino acid composition, tryptic peptide patterns, and the amino acid sequence of the NH₂-terminal segment. The oxygen equilibrium of a dimeric fraction has been determined at pH 7.5 and 20 °C; the pressure of half-saturation is 2.3 mm Hg.     

Specific Optical Rotations and the Horeau Effect

The observation of nonequivalence of optical and enantiomeric purities, referred to as the Horeau effect, is thought to arise from molecular aggregation in liquid solutions. Although this effect was first observed in 1969, the conditions under which this effect may, or may not, be observable are not established. Considering the formation of dimers as the simplest form of aggregation, the expressions for specific optical rotations in the presence of homochiral and heterochiral monomer–dimer equilibria are presented. Analysis of these equations indicates that the Horeau effect will not be observable even in the presence of aggregation under either of the following two situations: 1) The specific optical rotation of the monomeric species is equal to that of the dimeric species; 2) The heterochiral equilibrium constant is twice that of the homochiral equilibrium constant. Chirality 28:181–185, 2016. © 2015 Wiley Periodicals, Inc.     

Effect of zinc binding on β-amyloid structure and dynamics: implications for Aβ aggregation

Assembly of β-amyloid (Aβ) peptide into toxic oligomers is widely believed to initiate Alzheimer's disease pathogenesis. Under in vitro physiological conditions, zinc (Zn(II)) can bind to Aβ and redirect its assembly from amyloid fibrillar toward less toxic amorphous aggregation. Propensity of Aβ to go toward a specific form of aggregate state is determined by structural and dynamical properties of the initial monomeric as well as the aggregate state. Here we probe the structural and dynamical impact of binding of Zn(II) to monomeric Aβ40 using NMR spectroscopy. To obtain further support for the importance of intrinsic dynamics in the aggregation precursor, ¹⁵N relaxation measurements were also performed for Aβ42, the more fibrillar aggregation-prone variant of Aβ. The combined data suggest that, upon Zn(II)-binding to the N-terminus of Aβ40, a relatively rigid turnlike structure is induced at residues Val²⁴-Lys²⁸ whereas the residues flanking this region become more mobile on the picosecond-to-nanosecond timescale. This is in contrast to the increased rigidity of Aβ42 at the C-terminus, and proposed to be linked to the higher propensity of Zn(II)-bound peptide to form amorphous aggregates with less entropic penalties than their fibrillar counterparts.     

Antiplasmodial activity study of angiotensin II via Ala scan analogs

Angiotensin II (AII) as well as analog peptides shows antimalarial activity against Plasmodium gallinaceum and Plasmodium falciparum, but the exact mechanism of action is still unknown. This work presents the solid‐phase synthesis and characterization of eight peptides corresponding to the alanine scanning series of AII plus the amide‐capped derivative and the evaluation of the antiplasmodial activity of these peptides against mature P. gallinaceum sporozoites. The Ala screening data indicates that the replacement of either the Ile⁵ or the His⁶ residues causes minor effects on the in vitro antiplasmodial activity compared with AII, i.e. AII (88%), [Ala⁶]‐AII (79%), and [Ala⁵]‐AII (75%). Analogs [Ala³]‐AII, [Ala¹]‐AII, and AII‐NH₂ showed antiplasmodial activity around 65%, whereas the activity of the [Ala⁸]‐AII, [Ala⁷]‐AII, [Ala⁴]‐AII, and [Ala²]‐AII analogs is lower than 45%. Circular dichroism data suggest that AII and the most active analogs adopt a β‐fold conformation in different solutions. All AII analogs, except [Ala⁴]‐AII and [Ala⁸]‐AII, show contractile responses and interact with the AT₁ receptor, [Ala⁵]‐AII and [Ala⁶]‐AII. In conclusion, this approach is helpful to understand the contribution of each amino acid residue to the bioactivity of AII, opening new perspectives toward the design of new sporozoiticidal compounds. Copyright © 2014 European Peptide Society and John Wiley & Sons, Ltd.     

Basic polypeptides as histone models. Synthesis, conformation, and interaction with DNA of statistical copolymers (Lys x,Ala y)n.

Statistical copolymers (Lys^x,Ala^y)_n were synthesized by copolymerization of N-carboxyanhydrides of L -amino acids. The conformation of copolymers in aqueous solutions was investigated using circular dichroism (CD). Calculations based on the CD data showed that polymers (Lys^x,Ala^y)_n can exhibit a random conformation, an α-helix, and a β-structure in various ratios. CD spectra of complexes of copolymers with DNA prepared by gradual dialysis from a high ionic strength to 0.15 M NaCl can be correlated with the copolymer conformation in medium and high ionic strength. For copolymers forming an α-helix and β-structure, these spectra show resemblance with similar spectra of complexes of those histones that are able to exhibit ordered conformations.     

Generation and quenching of singlet molecular oxygen by aggregated bacteriochlorophyll d in model systems and chlorosomes

Both photogeneration and quenching of singlet oxygen by monomeric and aggregated (dimeric and oligomeric) molecules of bacteriochlorophyll (BChl) d have been studied in solution and in chlorosomes isolated from the green photosynthetic bacterium Chlorobium vibrioforme f. thiosulfatophilum. The yield of singlet-oxygen photogeneration by pigment dimers was about 6 times less than for monomers. Singlet oxygen formation was not observed in oligomer-containing solutions or in chlorosomes. To estimate the efficiency of singlet oxygen quenching an effective rate constant for ¹O₂ quenching by BChl molecules (k_q ^M) was determined using the Stern-Volmer equation and the total concentration of BChl d in the samples. In solutions containing only monomeric BChl, the k_q ^M values coincide with the real values for ¹O₂ quenching rate constants by BChl molecules. Aggregation weakly influenced the k_q ^M values in pigment solutions. In chlorosomes (which contain both BChl and carotenoids) the k_q ^M value was less than in solutions of BChl alone and much less than in acetone extracts from chlorosomes. Thus ¹O₂ quenching by BChl and carotenoids is much less efficient in chlorosomes than in solution and is likely caused primarily by BChl molecules which are close to the surface of the large chlorosome particles. The data allow a general conclusion that monomeric and dimeric chlorophyll molecules are the most likely sources of ¹O₂ formation in photosynthetic systems and excitation energy trapping by the long wavelength aggregates as well as ¹O₂ physical quenching by monomeric and aggregated chlorophyll can be considered as parts of the protective system against singlet oxygen formation.Abbreviations BChl bacteriochlorophyll - MBpd methyl bacteriopheophorbide - Chl chlorophyll - TPP meso-tetraphenylporphyrin - TPPS meso-tetra (p-sulfophenyl) porphyrin     

A correlation between differential structural features and the degree of endopeptidase activity of type A botulinum neurotoxin in aqueous solution

Botulinum neurotoxin type A is one of the most toxic substances known to man (LD(50) for mouse 0.1 ng/kg). It is also an effective therapeutic drug against involuntary muscle disorders and for pain management. BoNT/A is a Zn(2+) endopeptidase which selectively cleaves SNAP-25 (synaptosomal-associated protein of 25 kDa), a critical component of the exocytotic machinery. Based on nucleotide sequence, BoNT/A is a 145 kDa protein, which appears as a 145 kDa protein band on sodium dodecyl sulfate--polyacrylamide gel electrophoresis. We have examined the structure of BoNT/A in aqueous solution, and found the structure in aqueous solution differs dramatically from that resolved by X-ray crystallography, both at secondary and at quaternary levels. In terms of secondary structure, BoNT/A in aqueous solution has about 47% beta-sheet structure as revealed by infrared spectroscopy, while X-ray crystallography revealed only 17% beta-sheet structure. In terms of quaternary structure, the estimated molecular mass of the native BoNT/A in aqueous solution ranged between 230 and 314 kDa, based on results from different chemical and biophysical techniques (native gel electrophoresis, chemical cross-linking, size exclusion chromatography, and fluorescence anisotropy). These results indicate that BoNT/A exists as a dimer in aqueous solution, which contrasts with the reported monomeric structure of BoNT/A based on X-ray crystallography. The dimeric form of BoNT/A can self-dissociate into the monomeric form at a concentration lower than 50 nM. This concentration-dependent structural change has a significant impact on the endopeptidase activity of BoNT/A: the catalytic efficiency of the monomeric BoNT/A is about 4-fold higher than that of its dimeric form. This difference implies a sterically restricted catalytic site of BoNT/A in the dimeric form of BoNT/A.     

Effects of Group 3 LEA protein model peptides on desiccation-induced protein aggregation

Group 3 late embryogenesis abundant (G3LEA) proteins have amino acid sequences with characteristic 11-mer motifs and are known to reduce aggregation of proteins during dehydration. Previously, we clarified the structural and thermodynamic properties of the 11-mer repeating units in G3LEA proteins using synthetic peptides composed of two or four tandem repeats originating from an insect (Polypedilum vanderplanki), nematodes and plants. The purpose of the present study is to test the utility of such 22-mer peptides as protective reagents for aggregation-prone proteins. For lysozyme, desiccation-induced aggregation was abrogated by low molar ratios of a 22-mer peptide, PvLEA-22, derived from a P. vanderplanki G3LEA protein sequence. However, an unexpected behavior was noted for the milk protein, α-casein. On drying, the resultant aggregation was significantly suppressed in the presence of PvLEA-22 with its molar ratios>25 relative to α-casein. However, when the molar ratio was <10, aggregation occurred on addition of PvLEA-22 to aqueous solutions of α-casein. Other peptides derived from nematode, plant and randomized G3LEA protein sequences gave similar results. Such an anomalous solubility change in α-casein was shown to be due to a pH shift to ca. 4, a value nearly equal to the isoelectric point (pI) of α-casein, when any of the 22-mer peptides was mixed. These results demonstrate that synthetic peptides derived from G3LEA protein sequences can reduce protein aggregation caused both by desiccation and, at high molar ratios, also by pH effects, and therefore have potential as stabilization reagents.     

Structure of the monomeric isocitrate dehydrogenase: evidence of a protein monomerization by a domain duplication

NADP(+)-dependent isocitrate dehydrogenase is a member of the beta-decarboxylating dehydrogenase family and catalyzes the oxidative decarboxylation reaction from 2R,3S-isocitrate to yield 2-oxoglutarate and CO(2) in the Krebs cycle. Although most prokaryotic NADP(+)-dependent isocitrate dehydrogenases (IDHs) are homodimeric enzymes, the monomeric IDH with a molecular weight of 80-100 kDa has been found in a few species of bacteria. The 1.95 A crystal structure of the monomeric IDH revealed that it consists of two distinct domains, and its folding topology is related to the dimeric IDH. The structure of the large domain repeats a motif observed in the dimeric IDH. Such a fusional structure by domain duplication enables a single polypeptide chain to form a structure at the catalytic site that is homologous to the dimeric IDH, the catalytic site of which is located at the interface of two identical subunits.     

Flexibility in HIV-1 assembly subunits: solution structure of the monomeric C-terminal domain of the capsid protein

The protein CA forms the mature capsid of human immunodeficiency virus. Hexamerization of the N-terminal domain and dimerization of the C-terminal domain, CAC, occur during capsid assembly, and both domains constitute potential targets for anti-HIV inhibitors. CAC homodimerization occurs mainly through its second helix, and is abolished when its sole tryptophan is mutated to alanine. Previous thermodynamic data obtained with the dimeric and monomeric forms of CAC indicate that the structure of the mutant resembles that of a monomeric intermediate found in the folding and association reactions of CAC. We have solved the three-dimensional structure in aqueous solution of the monomeric mutant. The structure is similar to that of the subunits in the dimeric, nonmutated CAC, except the segment corresponding to the second helix, which is highly dynamic. At the end of this region, the polypeptide chain is bent to bury several hydrophobic residues and, as a consequence, the last two helices are rotated 90 degrees when compared to their position in dimeric CAC. The previously obtained thermodynamic data are consistent with the determined structure of the monomeric mutant. This extraordinary ability of CAC to change its structure may contribute to the different modes of association of CA during HIV assembly, and should be taken into account in the design of new drugs against this virus.     

NMR of alpha-synuclein-polyamine complexes elucidates the mechanism and kinetics of induced aggregation

The aggregation of alpha-synuclein is characteristic of Parkinson's disease (PD) and other neurodegenerative synucleinopathies. The 140-aa protein is natively unstructured; thus, ligands binding to the monomeric form are of therapeutic interest. Biogenic polyamines promote the aggregation of alpha-synuclein and may constitute endogenous agents modulating the pathogenesis of PD. We characterized the complexes of natural and synthetic polyamines with alpha-synuclein by NMR and assigned the binding site to C-terminal residues 109-140. Dissociation constants were derived from chemical shift perturbations. Greater polyamine charge (+2 --> +5) correlated with increased affinity and enhancement of fibrillation, for which we propose a simple kinetic mechanism involving a dimeric nucleation center. According to the analysis, polyamines increase the extent of nucleation by approximately 10(4) and the rate of monomer addition approximately 40-fold. Significant secondary structure is not induced in monomeric alpha-synuclein by polyamines at 15 degrees C. Instead, NMR reveals changes in a region (aa 22-93) far removed from the polyamine binding site and presumed to adopt the beta-sheet conformation characteristic of fibrillar alpha-synuclein. We conclude that the C-terminal domain acts as a regulator of alpha-synuclein aggregation.