Poly-(L-alanine) expansions form core beta-sheets that nucleate amyloid assembly

Expansion to a total of 11-17 sequential alanine residues from the normal number of 10 in the polyadenine-binding protein nuclear-1 (PABPN1) results in formation of intranuclear, fibrillar inclusions in skeletal muscle and hypothalamic neurons in adult-onset, dominantly inherited oculopharyngeal muscular dystrophy (OPMD). To understand the role that homopolymeric length may play in the protein misfolding that leads to the inclusions, we analyzed the self-assembly of synthetic poly-(L-alanine) peptides having 3-20 residues. We found that the conformational transition and structure of polyalanine (polyAla) assemblies in solution are not only length-dependent but also are determined by concentration, temperature, and incubation time. No beta-sheet complex was detected for those peptides characterized by n < 8, where n is number of alanine residues. A second group of peptides with 7 < n < 15 showed varying levels of complex formation, while for those peptides having n > 15, the interconversion process from the monomeric to the beta-sheet complex was complete under any of the tested experimental conditions. Unlike the typical tinctorial properties of amyloid fibrils, polyalanine fibrils did not show fluorescence with thioflavin T or apple-green birefringence with Congo red; however, like amyloid, X-ray diffraction showed that the peptide chains in these fibrils were oriented normal to the fibril axis (i.e., in the cross-beta arrangement). Neighboring beta-sheets are quarter-staggered in the hydrogen-bonding direction such that the alanine side-chains were closely packed in the intersheet space. Strong van der Waals contacts between side-chains in this arrangement likely account for the high stability of the macromolecular fibrillar complex in solution over a wide range of temperature (5-85 degrees C), and pH (2-10.5), and its resistance to denaturant (< 8 M urea) and to proteases (protease K, trypsin). We postulate that a similar stabilization of an expanded polyalanine stretch could form a core beta-sheet structure that mediates the intermolecular association of mutant proteins into fibrillar inclusions in human pathologies.     

A distinct 14 residue site triggers coiled-coil formation in cortexillin I.

We have investigated the process of the assembly of the Dictyostelium discoideum cortexillin I oligomerization domain (Ir) into a tightly packed, two-stranded, parallel coiled-coil structure using a variety of recombinant polypeptide chain fragments. The structures of these Ir fragments were analyzed by circular dichroism spectroscopy, analytical ultracentrifugation and electron microscopy. Deletion mapping identified a distinct 14 residue site within the Ir coiled coil, Arg311-Asp324, which was absolutely necessary for dimer formation, indicating that heptad repeats alone are not sufficient for stable coiled-coil formation. Moreover, deletion of the six N-terminal heptad repeats of Ir led to the formation of a four- rather than a two-helix structure, suggesting that the full-length cortexillin I coiled-coil domain behaves as a cooperative folding unit. Most interestingly, a 16 residue peptide containing the distinct coiled-coil 'trigger' site Arg311-Asp324 yielded approximately 30% helix formation as monomer, in aqueous solution. pH titration and NaCl screening experiments revealed that the peptide's helicity depends strongly on pH and ionic strength, indicating that electrostatic interactions by charged side chains within the peptide are critical in stabilizing its monomer helix. Taken together, these findings demonstrate that Arg311-Asp324 behaves as an autonomous helical folding unit and that this distinct Ir segment controls the process of coiled-coil formation of cortexillin I.     

Conformational properties of the beta(400-436) and beta(400-445) C-terminal peptides of porcine brain tubulin.

Two peptides from the C-terminal region of the major beta-tubulin isotype (400-436 and 400-445) that include the critical areas for interaction with MAP2 and tau were examined to determine their conformations in aqueous solution. Despite a high theoretical potential for alpha-helix formation, CD spectroscopy showed that these peptides consisted primarily of random coil with some reverse turn. This was unaffected by the presence of counterions to the negatively charged side chains (Ca2+, Mg2+), but did change when the side-chain charges were neutralized by lowering the pH; under these conditions, the alpha-helix content of the longer peptide rose to 25% and the C-terminal truncated peptide to 15%. The peptides also adopt alpha-helical structure in the presence of trifluoroethanol, the truncated peptide again attaining a lower maximum percentage. The beta(400-445) peptide was also studied by 1-D and 2-D NMR techniques. The results indicate that at pH 5.6 or 7 in an aqueous solution the peptide is extremely flexible and lacks regular secondary structure, consistent with the CD results. Both peptides inhibited microtubule-associated protein-stimulated tubulin assembly, with the longer peptide being about 4 times as inhibitory as the smaller peptide. Neither was inhibitory in the absence of microtubule-associated proteins, indicating that interaction with this species was necessary for inhibition. The greater activity of the longer peptide could be due to the extra negative charges in this peptide and/or the greater tendency of this peptide to form an alpha-helical structure under the appropriate conditions.     

Mechanism of energy coupling to entry and exit of neutral and branched chain amino acids in membrane vesicles of Streptococcus cremoris

The energetics of neutral and branched chain amino acid transport by membrane vesicles from Streptococcus cremoris have been studied with a novel model system in which beef heart mitochondrial cytochrome c oxidase functions as a proton-motive force (delta p) generating system. In the presence of reduced cytochrome c, a large delta p was generated with a maximum value at pH 6.0. Apparent H+/amino acid stoichiometries (napp) have been determined at external pH values between 5.5 and 8.0 from the steady state levels of accumulation and the delta p. For L-leucine napp (0.8) was nearly independent of the pH. For L-alanine and L-serine napp decreased from 0.9-1.0 at pH 5.5 to 0-0.2 at pH 8.0. The napp for the different amino acids decreased with increasing external amino acid concentration. At pH 6.0, first order rate constants for amino acid exit (kex) under steady state conditions for L-leucine, L-alanine, and L-serine were 1.1-1.3, 0.084, and 0.053 min-1, respectively. From the pH dependence of kex it is concluded that amino acid exit in steady state is the sum of two processes, pH-dependent carrier-mediated amino acid exit and pH-independent passive diffusion (external leak). The first order rate constant for passive diffusion increased with increasing hydrophobicity of the side chain of the amino acids. As a result of these processes the kinetic steady state attained is less than the amino acid accumulation ratio predicted by thermodynamic equilibrium. The napp determined from the steady state accumulation represents, therefore, a lower limit. It is concluded that the mechanistic stoichiometry (n) for L-leucine, L-alanine, and L-serine transport most likely equals 1.     

Self-assembly and polymerization of diacetylene-containing peptide amphiphiles in aqueous solution

A systematic study was performed of the fiber forming properties and polymerization characteristics of two peptide amphiphiles containing a diacetylene functionality in the alkyl tail comprising 23 and 25 C atoms, respectively. Both diyne containing peptide amphiphiles were able to form stable beta-sheet fibers of micrometers length in an aqueous solution. However, there was a large difference between the stability of the two amphiphiles. This was shown by a large difference in assembly and disassembly temperature and by different behavior during polymerization. Because the monomers were preorganized with a tight molecular packing, the polymerization could be carried out using wavelengths up to 532 nm. For both amphiphiles, the fiber structure did not change when the polymerization was carried out at an elevated temperature. The degree of polymerization, however, barely decreased for the longer amphiphile (2) but showed a gradual decline for the shorter one (1) when the temperature was raised from room temperature to the melting temperature of the fibers. Furthermore, the pH did not influence the fiber assembly for 2, but hampered it for 1 at alkaline pH. The fiber structure was, for both of the amphiphiles, not dependent on the pH. After polymerization, the molecular packing of the amphiphiles was only slightly influenced by an increase in temperature, as indicated by the small color change of polymerized fibers, which was also reversible. Additionally, pH had no influence on the assembly structure, as indicated by the color of the polymer which was the same at all pH values. Thus, both fibers increased in stability upon polymerization. The large difference in assembly and polymerization behavior of the two similar-looking amphiphiles 1 and 2, with a 23 or 25 carbon tail, is indicative of the subtlety of the assembly and disassembly processes in these fibrous architectures.     

An investigation of human oxyhemoglobin beta tetramer dissociation using haptoglobin binding

Haptoglobin was used as a macromolecular probe to investigate the formation of human oxyhemoglobin beta chain dimers from tetramers in 0.1 M potassium phosphate buffer, 20 degrees C at pH 7 and pH 8. Monitoring of spectral changes upon mixing haptoglobin with beta heme chains (2.5 and 5 micromolar) revealed an overall decrease in absorbance accompanied by a shift of the Soret spectral peak from 415 to 417 nm. The magnitude of the absorbance decrease was proportional to the beta concentration; the time courses consistently yielded greater color at pH 8 than at pH 7. At pH 8, two exponential phases of 0.47 min-1 and 0.084 min-1 were seen whose rates remained invariant with concentration. In contrast, only one exponential process was evident at pH 7, yielding a first order rate constant of 0.21 min-1. We have spectrophotometrically followed the beta chain tetramer to dimer dissociation reaction, thus providing information about the contribution of this step to hemoglobin assembly.     

Mycolic acid-containing glycolipid as a possible virulence factor of Rhodococcus equi for mice

By the use of various Rhodococcus equi strains differing in the length of carbon chains of glycolipid, we examined whether the glycolipid, glucose monomycolate, was contributing to the virulence of R. equi for mice. R. equi strains with longer carbon chain mycolic acid showed a higher virulence as determined by lethality and granuloma formation in mice than those with shorter ones. When purified glycolipid was injected into mice, granuloma formation and liver damage were most prominent with the glycolipid having longer carbon chain mycolic acid. Only a representative strain with longer carbon chain mycolic acid persisted in the spleen of mice after intravenous injection, while a strain with shorter carbon chain mycolic acid was readily eliminated. These results suggested that glycolipid was at least one of the virulence factors of R. equi and that the carbon chain length of mycolic acid might be critical in the expression of virulence.     

Molecular dynamics study of polyethylene chain non-isothermal crystallisation: effects of chain length and branch structure

The molecular dynamics simulations in this work were aimed to provide a molecular insight into chain structure effects on non-isothermal crystallisation of polyethylene (PE) chains. The crystallisation behaviours were influenced by chain length and cooling rate in linear PE chain crystallisation: C100 and C150 were unable to fold into crystals. From C1000 to C3000, crystallisation abilities became stronger as chain length increased. From C5000 to C14000, chain length had no influence on crystallisation abilities. Final morphologies changed from rotator phase to single crystal domain, and to multi crystal domains as chain length increased. The formation of multi crystal domains with longer chain was easier than with the shorter chain in identical conditions. Branch content influenced not only the crystallisation kinetics but also final morphologies in non-isothermal crystallisation. The branches were defective in nucleation process, which was reflected in the crystal growth process. Crystallisation temperature, rate and crystallinity decreased, and the morphologies became disordered as branch content increased. Changes of final morphologies from single crystal domain to multi crystal domains were found under the influence of branch content and cooling rate. Trans-rich phenomenon was observed, and the trans-state population increment was prior to crystallinity increment. Crystallisation processes began at different crystallisation temperature when the trans-state populations reached a critical value which was independent of branch content.     

On the effect of surface active agents and their structure on the temperature-induced changes of normal and waxy wheat starch in aqueous suspension. Part II: A confocal laser scanning microscopy study

The location and penetration patterns of two fluorescently labelled, surface active molecules into normal and waxy wheat starch granules prior, during and after the temperature-induced gelatinization were studied by means of confocal laser scanning microscopy (CLSM). Amphiphilic dyes were found to have a tendency to penetrate wheat starch granules in aqueous suspension. The penetration patterns were however found to be dependent on the contact time, type of starch and the chain length (C₁₂ vs. C₁₆) of the amphiphilic dye. The penetration of amphiphilic dyes through the starch granule matrix proved to be less restricted in waxy than in normal wheat starch. For a given type of starch, the penetration of the longer chain dye was more constrained than that of the shorter chain one. The extent to which the dye diffuses into the granule matrix as it gelatinizes is also affected by the chain length of the dye, diffusion of the shorter chain dye occurring more profusely and at lower temperatures than for the longer chain one. These differences are suggested to be related to the dissociation temperature of the AM-amphiphilic dye complexes.     

Ion-pairing separation of bioactive peptides using an aqueous/octan-1-ol micro-extraction system from bovine haemoglobin complex hydrolysates

The ion-pair concept was applied on complex haemoglobin hydrolysates to extract two opioid peptides, LVV-haemorphin-7 and VV-haemorphin-7, in an aqueous/octan-1-ol micro-extraction system in the presence of alkyl-sulfonic acid as a surfactant agent and in relation to the haemorphin physico-chemical properties (charge, hydrophobicity). The effect of combined alkyl chain length/aqueous phase pH and the haem behaviour during the extraction, on the haemorphin recovery yield and enrichment has been determined. It has proved that transport over the organic phase is mediated by the alkyl-sulfonic acids, whatever be the aqueous phase pH. However, increasing both the alkyl chain length and the pH in the aqueous phase shows an haemorphin enrichment ratio increase but a recovery decrease of the extracted opioid peptides in the organic phase. Therefore, the best conditions to extract LVVh-7 and VVh-7 are the use of the octane-sulfonic acid at aqueous phase pH of 5 or 7 and the octane or the heptane-sulfonic acid with an aqueous phase pH of 5 or 7 respectively. In these conditions, a partition coefficient of 1.64 and 1.60 respectively for LVVh-7 and VVh-7 are obtained and represent about 40 times that acquired without agent.     

Cooperativity in macromolecular assembly

The thermodynamic principle of cooperativity is used to drive the formation of specific macromolecular complexes during the assembly of a macromolecular machine. Understanding cooperativity provides insight into the mechanisms that govern assembly and disassembly of multicomponent complexes. Our understanding of assembly mechanisms is lagging considerably behind our understanding of the structure and function of these complexes. A significant challenge remains in tackling the thermodynamics and kinetics of the intermolecular interactions required for all cellular functions.     

Effect of pH on complex formation between debranched waxy rice starch and fatty acids

Complex formations between debranched waxy rice starch (DBS) and fatty acids (FA) of different hydrocarbon chain lengths (8:0, 10:0, 12:0, 14:0, 16:0, and 18:0) were studied in an aqueous solution by measuring the blue colour stained with iodine. The objective of this study was to understand the effects of the solubility and hydrophobicity of guest molecules (FA) on the complex formation with DBS. Lauric acid (12:0) displayed the greatest complex forming ability with DBS by showing the least blue colour developed with iodine. The effect of pH (3-7) on the DBS/FA complex formation was evaluated by measuring the iodine-scanning spectra of the mixture. Short-chain FA (8:0) displayed less complex formation at pH>or=5, above the pK(a) of fatty acid (approximately 4.8), which suggested that the charge formation of the short-chain FA caused a lower partitioning of the FA into the hydrophobic cavity of the DBS single helix. On the contrary, FA of 10:0-18:0 displayed an increased complex formation at pH>5, which could be attributed to increased solubility of these longer-chain FA at a dissociated and ionized form. The hydrocarbon chain length of the FA had an important impact on the extent of the complex formation. A FA that had a shorter hydrocarbon chain was more soluble in an aqueous solution and more readily formed a complex with DBS. At pH 6 and 7 (above the pK(a)), 10:0 formed less inclusion complexes with DBS than did 12:0. Iodine-scanning spectra showed that the absorbances of all iodine-stained DBS/FA solutions at higher wavelength were substantially lower than that of the iodine-stained DBS alone, suggesting that FA preferentially formed inclusion complexes with DBS of longer chains.     

Protein interactions and misfolding analyzed by AFM force spectroscopy

Protein misfolding is conformational transition dramatically facilitating the assembly of protein molecules into aggregates of various morphologies. Spontaneous formation of specific aggregates, mostly amyloid fibrils, was initially believed to be limited to proteins involved in the development of amyloidoses. However, recent studies show that, depending on conditions, the majority of proteins undergo structural transitions leading to the appearance of amyloidogenic intermediates followed by aggregate formation. Various techniques have been used to characterize the protein misfolding facilitating the aggregation process, but no direct evidence as to how such a conformational transition increases the intermolecular interactions has been obtained as of yet. We have applied atomic force microscopy (AFM) to follow the interaction between protein molecules as a function of pH. These studies were performed for three unrelated and structurally distinctive proteins, alpha-synuclein, amyloid beta-peptide (Abeta) and lysozyme. It was shown that the attractive force between homologous protein molecules is minimal at physiological pH and increases dramatically at acidic pH. Moreover, the dependence of the pulling forces is sharp, suggesting a pH-dependent conformational transition within the protein. Parallel circular dichroism (CD) measurements performed for alpha-synuclein and Abeta revealed that the decrease in pH is accompanied by a sharp conformational transition from a random coil at neutral pH to the more ordered, predominantly beta-sheet, structure at low pH. Importantly, the pH ranges for these conformational transitions coincide with those of pulling forces changes detected by AFM. In addition, protein self-assembly into filamentous aggregates studied by AFM imaging was shown to be facilitated at pH values corresponding to the maximum of pulling forces. Overall, these results indicate that proteins at acidic pH undergo structural transition into conformations responsible for the dramatic increase in interprotein interaction and promoting the formation of protein aggregates.     

Thermodynamics of the hydrophobic effect. I. Coupling of aggregation and pK(a) shifts in solutions of aliphatic amines

Long aliphatic hydrocarbon chains aggregate in aqueous solution due to the hydrophobic effect, forming structures such as micelles and membranes, while amino groups titrate at basic pH. These two biologically important behaviors are linked in alkylamines, in which the pK(a) of the amino group is shifted downward by aggregation. In this paper we study the thermodynamics of these coupled processes, following aggregation by observing alkylamine pH titration behavior. The magnitude of the shift depended on the aliphatic chain length and on the concentration of alkylamine: longer chains and higher concentrations lowered the pK(a) to a greater extent. Gibbs free energies of protonation and aggregation were calculated from the pK(a) shifts. Enthalpies, entropies, and heat capacities were estimated by van't Hoff analysis from the pK(a) shift dependencies on temperature. However, the results were less precise than the calorimetrically measured values, as described in the following article. A model to calculate titration curves, pK(a) shifts, and aggregation of uncharged alkylamines as a function of aliphatic chain length, concentration, and temperature is presented.     

Interactions of voltage-sensing dyes with membranes. II. Spectrophotometric and electrical correlates of cyanine-dye adsorption to membranes.

The adsorption to bilayer membranes of the thiadicarbocyanine dyes, diSCn(5), has been studied as a function of the membrane's surface-charge density, the aqueous ionic strength, and the length (n) of the hydrocarbon side chain of the dye. "Probe" measurements in planar bilayers, microelectrophoresis of liposomes, and measurement of changes in dye absorbance and fluorescence in liposomes were used to study dye adsorption to membranes. These measurements indicated that the membrane:water partition coefficient for the dye monomer increases with the length of the hydrocarbon side chain. However, the formation of large aggregates in the aqueous phase also increases with increasing chain length and ionic strength so that the actual dye adsorbing to the membrane goes through a maximum at high but not at low ionic strengths. More dye adsorbs to negatively charged than neutral membranes. Membrane-bound dye spectra were easily resolved in negatively charged liposomes where it was observed that these dyes could exist as monomers, dimers, and large aggregates. For diSC1(5) a spectral peak was observed at low but not high ionic strengths (i.e. the conditions in which this dye appears to form voltage-gated channels) corresponding to small aggregates which appeared to adsorb to the membrane. Finally, the adsorption of these dyes to membranes results in more positive electrostatic potentials composed primarily of dye-induced "boundary" potentials and somewhat less of "double-layer" potentials.     

The catalytic core of an archaeal 2-oxoacid dehydrogenase multienzyme complex is a 42-mer protein assembly

The dihydrolipoyl acyl-transferase (E2) enzyme forms the structural and catalytic core of the tripartite 2-oxoacid dehydrogenase multienzyme complexes of the central metabolic pathways. Although this family of multienzyme complexes shares a common architecture, their E2 cores form homo-trimers that, depending on the source, further associate into either octahedral (24-mer) or icosahedral (60-mer) assemblies, as predicted by the principles of quasi-equivalence. In the crystal structure of the E2 core from Thermoplasma acidophilum, a thermophilic archaeon, the homo-trimers assemble into a unique 42-mer oblate spheroid. Analytical equilibrium centrifugation and small-angle X-ray scattering analyses confirm that this catalytically active 1.08 MDa assembly exists as a single species in solution, forming a hollow spheroid with a maximum diameter of 220 ?. In this paper we show that a monodisperse macromolecular assembly, built from identical subunits in non-identical environments, forms an irregular protein shell via non-equivalent interactions. This unusually irregular protein shell, combining cubic and dodecahedral geometrical elements, expands on the concept of quasi-equivalence as a basis for understanding macromolecular assemblies by showing that cubic point group symmetry is not a physical requirement in multienzyme assembly. These results extend our basic knowledge of protein assembly and greatly expand the number of possibilities to manipulate self-assembling biological complexes to be utilized in innovative nanotechnology applications.     

Effect of heavy chain phosphorylation on the polymerization and structure of Dictyostelium myosin filaments

In Dictyostelium amebas, myosin appears to be organized into filaments that relocalize during cell division and in response to stimulation by cAMP. To better understand the regulation of myosin assembly, we have studied the polymerization properties of purified Dictyostelium myosin. In 150 mM KCl, the myosin remained in the supernate following centrifugation at 100,000 g. Rotary shadowing showed that this soluble myosin was monomeric and that approximately 80% of the molecules had a single bend 98 nm from the head-tail junction. In very low concentrations of KCl (less than 10 mM) the Dictyostelium myosin was also soluble at 100,000 g. But rather than being monomeric, most of the molecules were associated into dimers or tetramers. At pH 7.5 in 50 mM KCl, dephosphorylated myosin polymerized into filaments whereas myosin phosphorylated to a level of 0.85 mol Pi/mol heavy chain failed to form filaments. The phosphorylated myosin could be induced to form filaments by lowering the pH or by increasing the magnesium concentration to 10 mM. The resulting filaments were bipolar, had blunt ends, and had a uniform length of approximately 0.43 micron. In contrast, filaments formed from fully dephosphorylated myosin were longer, had tapered ends, and aggregated to form very long, threadlike structures. The Dictyostelium myosin had a very low critical concentration for assembly of approximately 5 micrograms/ml, and this value did not appear to be affected by the level of heavy chain phosphorylation. The concentration of polymer at equilibrium, however, was significantly reduced, indicating that heavy chain phosphorylation inhibited the affinity of subunits for each other. Detailed assembly curves revealed that small changes in the concentration of KCl, magnesium, ATP, or H+ strongly influenced the degree of assembly. Thus, changes in both the intracellular milieu and the level of heavy chain phosphorylation may control the location and state of assembly of myosin in response to physiological stimuli.     

Fatty acid alcohol ester-synthesizing activity of lipoprotein lipase

The fatty acid alcohol ester-synthesizing activity of lipoprotein lipase (LPL) was characterized using bovine milk LPL. Synthesizing activities were determined in an aqueous medium using oleic acid or trioleylglycerol as the acyl donor and equimolar amounts of long-chain alcohols as the acyl acceptor. When oleic acid and hexadecanol emulsified with gum arabic were incubated with LPL, palmityl oleate was synthesized, in a time- and dose-dependent manner. Apo-very low density lipoprotein (apoVLDL) stimulated LPL-catalyzed palmityl oleate synthesis. The apparent equilibrium ratio of fatty acid alcohol ester/oleic acid was estimated using a high concentration of LPL and a long (20 h) incubation period. The equilibrium ratio was affected by the incubation pH and the alcohol chain length. When the incubation pH was below pH 7.0 and long chain fatty acyl alcohols were used as substrates, the fatty acid alcohol ester/free fatty acid equilibrium ratio favored ester formation, with an apparent equilibrium ratio of fatty acid alcohol ester/fatty acid of about 0.9/0.1. The equilibrium ratio decreased sharply at alkaline pH (above pH 8.0). The ratio also decreased when fatty alcohols with acyl chains shorter than dodecanol were used. When a trioleoylglycerol/fatty acyl alcohol emulsion was incubated with LPL, fatty acid alcohol esters were synthesized in a dose- and time-dependent fashion. Fatty acid alcohol esters were easily synthesized from trioleoylglycerol when fatty alcohols with acyl chains longer than dodecanol were used, but synthesis was decreased with fatty alcohols with acyl chain lengths shorter than decanol, and little synthesizing activity was detected with shorter-chain fatty alcohols such as butanol or ethanol.     

Nucleic acid binding-induced Gag dimerization in the assembly of Rous sarcoma virus particles in vitro

As also found for other retroviruses, the Rous sarcoma virus structural protein Gag is necessary and sufficient for formation of virus-like particles (VLPs). Purified polypeptide fragments comprising most of Gag spontaneously assemble in vitro at pH 6.5 into VLPs lacking a membrane, a process that requires nucleic acid. We showed previously that the minimum length of a DNA oligonucleotide that can support efficient assembly is 16 nucleotides (nt), twice the protein's binding site size. This observation suggests that the essential role of nucleic acid in assembly is to promote the formation of Gag dimers. In order to gain further insight into the role of dimerization, we have studied the assembly properties of two proteins, a nearly full-length Gag (deltaMBDdeltaPR) capable of proper in vitro assembly and a smaller Gag fragment (CTD-NC) capable of forming only irregular aggregates but with the same pH and oligonucleotide length requirements as for assembly with the larger protein. In analyses by sedimentation velocity and by cross-linking, both proteins remained monomeric in the absence of oligonucleotides or in the presence of an oligonucleotide of length 8 nt (GT8). At pH 8, which does not support assembly, binding to GT16 induced the formation of dimers of deltaMBDdeltaPR but not of CTD-NC, implying that dimerization requires the N-terminal domain of the capsid moiety of Gag. Assembly of VLPs was induced by shifting the pH of dimeric complexes of deltaMBDdeltaPR and GT16 from 8 to 6.5. An analogue of GT16 with a ribonucleotide linkage in the middle also supported dimer formation at pH 8. Even after quantitative cleavage of the oligonucleotide by treatment of the complex with RNase, these dimers could be triggered to undergo assembly by pH change. This result implies that protein-protein interactions stabilize the dimer. We propose that binding of two adjacent Gag molecules on a stretch of nucleic acid leads to protein-protein interactions that create a Gag dimer and that this species has an exposed surface not present in monomers which allows polymerization of the dimers into a spherical shell.     

Purification and properties of L-alanine aminotransferase from Chlamydomonas reinhardtii.

An enzyme which catalyzes the transamination of L-alanine with 2-oxoglutarate has been purified 157-fold to electrophoretic homogeneity from the unicellular green alga Chlamydomonas reinhardtii 6145c. The enzyme showed maximal activity at pH 7.3 and 50 degrees C, has an apparent molecular mass of 105 kDa as estimated by gel filtration, and consists of two identical subunits of 45 kDa each as deduced from PAGE/SDS studies. A stoichiometry of two moles pyridoxal 5-phosphate/mole enzyme was calculated. The enzyme has an isoelectric point of 8.3 and its absorption spectrum exhibits a maximum at 412 nm which is shifted to 330 nm upon addition of L-alanine. Pyridoxal 5-phosphate protected activity against heat inactivation and, to a minor extent, L-alanine and 2-oxoglutarate, but not L-glutamate. Spectral data and activity inhibition and protection studies strongly support the involvement of pyridoxal 5-phosphate in enzyme catalysis through a Schiff's base formation. The purified enzyme was able to transaminate only L-alanine and L-glutamate with glyoxylate out of ten amino acids tested. L-Alanine aminotransferase exhibited hyperbolic kinetic for 2-oxoglutarate, pyruvate, and L-glutamate, and nonhyperbolic behaviour for L-alanine. Apparent Km values were 0.054 mM for 2-oxoglutarate, 0.52 for L-glutamate, 0.24 mM for pyruvate, and 2.7 mM for L-alanine. Transamination of L-alanine in C. reinhardtii is a bisubstrate reaction with a bi-bi ping-pong mechanism, and is not inhibited by substrates.