Showing papers in "Biochemistry in 2002"

Biological and Chemical Applications of Fluorescence Correlation Spectroscopy: A Review†

Abstract: The mathematical concept of fluorescence correlation spectroscopy (FCS) 1 (1) emerged from quasi-elastic light scattering (QELS) spectroscopy ( 2) in the early 1970s. Compared to light scattering, the enhanced sensitivity of fluorescence to changes in molecular structure, chemistry, and local environment makes FCS a superior analytical tool for chemical kinetics studies ( 1, 3-5). The primary motivation for the invention of FCS was the study of chemical kinetics at very dilute concentrations in biological systems, such as the reversible binding reaction between ethidium bromide, a fluorescent nucleic acid synthesis inhibitor, and DNA ( 1). Theoretical and experimental studies ( 3, 4, 6) soon established that FCS could measure not only diffusion coefficients but also chemical rate constants, concentration, aggregation, and rotational dynamics ( 3-9). Building on this foundation, significant advances in the understanding of lipid diffusion in membranes were made soon after the birth of FCS ( 10) using a confocal microscope geometry ( 11) introduced into FCS by Koppel et al. ( 7) and still used today. Recently, technological advances in detectors, autocorrelation electronics, and confocal microscopy were incorporated into FCS, mainly in the laboratories of R. Rigler and M. Eigen (12, 13). A detailed theoretical framework on the effects of translational and rotational motion of a fluorescent molecule undergoing chemical reactions, in a three-dimensional (3D) Gaussian observation volume, has been introduced ( 14). Statistical analysis provided the basis for optimizing the signal-to-noise ratio (S/N) in FCS ( 15). Furthermore, analytic functions describing molecular translation (16), shot noise effects on higher-order fluorescence fluctuation moments ( 17), and the effect of the observation volume (18) on the S/N were explored. Finally, the ability of FCS to resolve multiple species with equivalent diffusion properties was extended by probability analysis of fluorescence intensity distributions ( 19-22). These advances extend the horizon of FCS in biological and chemical studies (for a recent review, see ref 23).

Mapping protein-protein interactions in solution by NMR spectroscopy.

Abstract: NMR is very well suited to the study of especially weak protein-protein interactions, as no crystallization is required. The available NMR methods to this end are reviewed and illustrated with applications from the recent biochemical literature: intermolecular NOEs, cross-saturation, chemical shift perturbation, dynamics and exchange perturbation, paramagnetic methods, and dipolar orientation. Most of these methods are now routinely applied for complexes with total molecular mass of 60 kDa and can likely be applied to systems up to 1000 kDa. A substantial fraction of complexes studied show distinct effects of induced fit affecting structural and dynamical properties beyond the contact interface.

Functional consequences of cysteine modification in the ligand binding sites of peroxisome proliferator activated receptors by GW9662.

Abstract: In the course of a high throughput screen to search for ligands of peroxisome proliferator activated receptor-γ (PPARγ), we identified GW9662 using a competition binding assay against the human ligand binding domain. GW9662 had nanomolar IC50 versus PPARγ and was 10- and 600-fold less potent in binding experiments using PPARα and PPARδ, respectively. Pretreatment of all three PPARs with GW9662 resulted in the irreversible loss of ligand binding as assessed by scintillation proximity assay. Incubation of PPAR with GW9662 resulted in a change in the absorbance spectra of the receptors consistent with covalent modification. Mass spectrometric analysis of the PPARγ ligand binding domain treated with GW9662 established Cys285 as the site of covalent modification. This cysteine is conserved among all three PPARs. In cell-based reporter assays, GW9662 was a potent and selective antagonist of full-length PPARγ. The functional activity of GW9662 as an antagonist of PPARγ was confirmed in an assay of adipocyte diff...

Vesicle Permeabilization by Protofibrillar α-Synuclein Is Sensitive to Parkinson's Disease-Linked Mutations and Occurs by a Pore-like Mechanism†

Abstract: Two mutations in the protein α-synuclein (A30P and A53T) are linked to an autosomal dominant form of Parkinson's disease. Both mutations accelerate the formation of prefibrillar oligomers (protofibrils) in vitro, but the mechanism by which they promote toxicity is unknown. Protofibrils of wild-type α-synuclein bind and permeabilize acidic phospholipid vesicles. This study examines the relative membrane permeabilizing activities of the wild type, mutant, and mouse variants of protofibrillar α-synuclein and the mechanism of membrane permeabilization. Protofibrillar A30P, A53T, and mouse variants were each found to have greater permeabilizing activities per mole than the wild-type protein. The leakage of vesicular contents induced by protofibrillar α-synuclein exhibits a strong preference for low-molecular mass molecules, suggesting a pore-like mechanism for permeabilization. Under conditions in which the vesicular membrane is less stable (lack of calcium as a phospholipid counterion), protofibril permeabili...

Lipid rafts are enriched in arachidonic acid and plasmenylethanolamine and their composition is independent of caveolin-1 expression: a quantitative electrospray ionization/mass spectrometric analysis.

Abstract: Lipid rafts are specialized cholesterol-enriched membrane domains that participate in cellular signaling processes. Caveolae are related domains that become invaginated due to the presence of the structural protein, caveolin-1. In this paper, we use electrospray ionization mass spectrometry (ESI/MS) to quantitatively compare the phospholipids present in plasma membranes and nondetergent lipid rafts from caveolin-1-expressing and nonexpressing cells. Lipid rafts are enriched in cholesterol and sphingomyelin as compared to the plasma membrane fraction. Expression of caveolin-1 increases the amount of cholesterol recovered in the lipid raft fraction but does not affect the relative proportions of the various phospholipid classes. Surprisingly, ESI/MS demonstrated that lipid rafts are enriched in plasmenylethanolamines, particularly those containing arachidonic acid. While the total content of anionic phospholipids was similar in plasma membranes and nondetergent lipid rafts, the latter were highly enriched in phosphatidylserine but relatively depleted in phosphatidylinositol. Detergent-resistant membranes made from the same cells showed a higher cholesterol content than nondetergent lipid rafts but were depleted in anionic phospholipids. In addition, these detergent-resistant membranes were not enriched in arachidonic acid-containing ethanolamine plasmalogens. These data provide insight into the structure of lipid rafts and identify potential new roles for these domains in signal transduction.

Crystal structure of a four-copper laccase complexed with an arylamine: insights into substrate recognition and correlation with kinetics.

Abstract: Laccases are multicopper oxidases that catalyze the oxidation of a wide range of phenols or arylamines, and their use in industrial oxidative processes is increasing. We purified from the white rot fungus Trametes versicolor a laccase that exists as five different isozymes, depending on glycosylation. The 2.4 A resolution structure of the most abundant isozyme of the glycosylated enzyme was solved. The four copper atoms are present, and it is the first crystal structure of a laccase in its active form. The crystallized enzyme binds 2,5-xylidine, which was used as a laccase inducer in the fungus culture. This arylamine is a very weak reducing substrate of the enzyme. The cavity enclosing 2,5-xylidine is rather wide, allowing the accommodation of substrates of various sizes. Several amino acid residues make hydrophobic interactions with the aromatic ring of the ligand. In addition, two charged or polar residues interact with its amino group. The first one is an histidine that also coordinates the copper that functions as the primary electron acceptor. The second is an aspartate conserved among fungal laccases. The purified enzyme can oxidize various hydroxylated compounds of the phenylurea family of herbicides that we synthesized. These phenolic substrates have better affinities at pH 5 than at pH 3, which could be related to the 2,5-xylidine binding by the aspartate. This is the first high-resolution structure of a multicopper oxidase complexed to a reducing substrate. It provides a model for engineering laccases that are either more efficient or with a wider substrate specificity.

Proline Cis-Trans Isomerization and Protein Folding †

Abstract: Proline cis−trans isomerization plays a key role in the rate-determining steps of protein folding. The energetic origin of this isomerization process is summarized, and the folding and unfolding of disulfide-intact bovine pancreatic ribonuclease A is used as an example to illustrate the kinetics and structural features of conformational changes from the heterogeneous unfolded state (consisting of cis and trans isomers of X-Pro peptide groups) to the native structure in which only one set of proline isomers is present.

Phosphorylation of p47phox sites by PKC alpha, beta II, delta, and zeta: effect on binding to p22phox and on NADPH oxidase activation.

Abstract: Production of superoxide anions by the multicomponent enzyme of human neutrophil NADPH oxidase is accompanied by extensive phosphorylation of p47(phox), one of its cytosolic components. p47(phox) is an excellent substrate for protein kinase C (PKC), but the respective contribution of each PKC isoform to this process is not clearly defined. In this study, we found that PKC isoforms known to be present in human neutrophils (PKC alpha, beta, delta, and zeta) phosphorylate p47(phox) in a time- and concentration-dependent manner, with apparent K(m) values of 10.33, 3.37, 2.37, and 2.13 microM for PKC alpha, beta II, delta, and zeta, respectively. Phosphopeptide mapping of p47(phox) showed that, as opposed to PKC zeta, PKC alpha, beta II, and delta are able to phosphorylate all the major PKC sites. The use of p47(phox) mutants identified serines 303, 304, 315, 320, 328, 359, 370, and 379 as targets of PKC alpha, beta II, and delta. Comparison of the intensity of phosphopeptides suggests that Ser 328 is the most phosphorylated serine. The ability of each PKC isoform to induce p47(phox) to associate with p22(phox) was tested by using an overlay technique; the results showed that all the PKC isoforms that were studied induce p47(phox) binding to the cytosolic fragment of p22(phox). In addition, PKC alpha, beta II, delta, and zeta were able to induce production of superoxide anions in a cell-free system using recombinant cytosolic proteins. Surprisingly, PKC zeta, which phosphorylates a subset of selective p47(phox) sites, induced stronger activation of the NADPH oxidase. Taken together, these results suggest that PKC alpha, beta II, delta, and zeta expressed in human neutrophils can individually phosphorylate p47(phox) and induce both its translocation and NADPH oxidase activation. In addition, phosphorylation of some serines could have an inhibitory effect on oxidase activation.

Novel antisense and peptide nucleic acid strategies for controlling gene expression.

Abstract: Antisense oligonucleotides have the potential to make revolutionary contributions to basic science and medicine. Oligonucleotides can bind mRNA and inhibit translation. Because they can be rapidly synthesized to be complementary to any sequence, they offer ideal tools for exploiting the massive amount of genome information now available. However, until recently, this potential was largely theoretical, and antisense experiments often produced inconclusive or misleading outcomes. This review will discuss the chemical and biological properties of some of the different types of oligomers now available and describe the challenges confronting in vitro and in vivo use of oligonucleotides. Oligomers with improved chemical properties, combined with advances in cell biology and success in clinical trials, are affording powerful new options for basic research, biotechnology, and medicine.

Molecular Features of the Copper Binding Sites in the Octarepeat Domain of the Prion Protein

Abstract: Recent evidence suggests that the prion protein (PrP) is a copper binding protein. The N-terminal region of human PrP contains four sequential copies of the highly conserved octarepeat sequence PHGGGWGQ spanning residues 60-91. This region selectively binds Cu2+ in vivo. In a previous study using peptide design, EPR, and CD spectroscopy, we showed that the HGGGW segment within each octarepeat comprises the fundamental Cu2+ binding unit [Aronoff-Spencer et al. (2000) Biochemistry 40, 13760-13771]. Here we present the first atomic resolution view of the copper binding site within an octarepeat. The crystal structure of HGGGW in a complex with Cu2+ reveals equatorial coordination by the histidine imidazole, two deprotonated glycine amides, and a glycine carbonyl, along with an axial water bridging to the Trp indole. Companion S-band EPR, X-band ESEEM, and HYSCORE experiments performed on a library of 15N-labeled peptides indicate that the structure of the copper binding site in HGGGW and PHGGGWGQ in solution is consistent with that of the crystal structure. Moreover, EPR performed on PrP(23-28, 57-91) and an 15N-labeled analogue demonstrates that the identified structure is maintained in the full PrP octarepeat domain. It has been shown that copper stimulates PrP endocytosis. The identified Gly-Cu linkage is unstable below pH approximately 6.5 and thus suggests a pH-dependent molecular mechanism by which PrP detects Cu2+ in the extracellular matrix or releases PrP-bound Cu2+ within the endosome. The structure also reveals an unusual complementary interaction between copper-structured HGGGW units that may facilitate molecular recognition between prion proteins, thereby suggesting a mechanism for transmembrane signaling and perhaps conversion to the pathogenic form.

DNA Nanoparticles and Development of DNA Delivery Vehicles for Gene Therapy

Abstract: DNA transport through the cell membrane is an essential requirement for gene therapy, which utilizes oligonucleotides and plasmid DNA. However, membrane transport of DNA is an inefficient process, and the mechanism(s) by which this process occurs is not clear. Although viral vectors are effective in gene therapy, the immune response elicited by viral proteins poses a major problem. Therefore, several laboratories are involved in the development of nonviral DNA delivery vehicles. These vehicles include polyamines, polycationic lipids, and neutral polymers, capable of condensing DNA to nanoparticles with radii of 20−100 nm. Although the structural and energetic forces involved in DNA condensation have been studied by physical biochemists for the past 25 years, this area has experienced a resurgence of interest in recent years because of the influx of biotechnologists involved in developing gene therapy protocols to combat a variety of human diseases. Despite an intense effort to study the mechanism(s) of DN...

A Novel ε-Cleavage within the Transmembrane Domain of the Alzheimer Amyloid Precursor Protein Demonstrates Homology with Notch Processing†

Abstract: Proteolytic processing of the transmembrane domain of the amyloid precursor protein (APP) is a key component of Alzheimer's disease pathogenesis. Using C-terminally tagged APP derivatives, we have identified by amino-terminal sequencing a novel cleavage site of APP, at Leu-49, distal to the γ-secretase site. This was termed e-cleavage. Brefeldin A treatment and pulse−chase experiments indicate that this cleavage occurs late in the secretory pathway. The level of e-cleavage is decreased by expression of presenilin-1 mutants known to impair Aβ formation, and it is sensitive to the γ-secretase inhibitors MDL28170 and L-685,458. Remarkably, it shares similarities with site 3 cleavage of Notch-1: membrane topology, cleavage before a valine, dependence on presenilins, and inhibition profile.

Annular α-Synuclein Protofibrils Are Produced When Spherical Protofibrils Are Incubated in Solution or Bound to Brain-Derived Membranes†

Abstract: The Parkinson's disease substantia nigra is characterized by the loss of dopaminergic neurons and the presence of cytoplasmic fibrillar Lewy bodies in surviving neurons. The major fibrillar protein of Lewy bodies is α-synuclein. Two point mutations in the α-synuclein gene are associated with autosomal-dominant Parkinson's disease (FPD). Studies of the in vitro fibrillization behavior of the mutant proteins suggest that fibril precursors, or α-synuclein protofibrils, rather than the fibrils, may be pathogenic. Atomic force microscopy (AFM) revealed two distinct forms of protofibrillar α-synuclein: rapidly formed spherical protofibrils and annular protofibrils, which were produced on prolonged incubation of spheres. The spherical protofibrils bound to brain-derived membrane fractions much more tightly than did monomeric or fibrillar α-synuclein, and membrane-associated annular protofibrils were observed. The structural features of α-synuclein annular protofibrils are reminiscent of bacterial pore-forming t...

Interactions of Amyloid β-Protein with Various Gangliosides in Raft-Like Membranes: Importance of GM1 Ganglioside-Bound Form as an Endogenous Seed for Alzheimer Amyloid†

Abstract: GM1 ganglioside-bound amyloid β-protein (GM1-Aβ), found in brains exhibiting early pathological changes of Alzheimer's disease (AD) plaques, has been suggested to accelerate amyloid fibril formatio...

Weak protein-protein interactions are sufficient to drive assembly of hepatitis B virus capsids.

Abstract: Hepatitis B virus (HBV) is an enveloped DNA virus with a spherical capsid (or core). The capsid is constructed from 120 copies of the homodimeric capsid protein arranged with T = 4 icosahedral symmetry. We examined in vitro assembly of purified E. coli expressed HBV capsid protein. After equilibration, concentrations of capsid and dimer were evaluated by size exclusion chromatography. The extent of assembly increased as temperature and ionic strength increased. The concentration dependence of capsid assembly conformed to the equilibrium expression: Kcapsid = [capsid]/[dimer]120. Given the known geometry for HBV capsids and dimers, the per capsid assembly energy was partitioned into energy per subunit−subunit contact. We were able to make three major conclusions. (i) Weak interactions (from −2.9 kcal/mol at 21 °C in low salt to −4.4 kcal/mol at 37 °C in high salt) at each intersubunit contact result in a globally stable capsid; weak intersubunit interactions may be the basis for the phenomenon of capsid b...

Lipids in the Structure, Folding, and Function of the KcsA K+ Channel†

Abstract: Lipid molecules surround an ion channel in its native environment of cellular membranes. The importance of the lipid bilayer and the role of lipid protein interactions in ion channel structure and function are not well understood. Here we demonstrate that the bacterial potassium channel KcsA binds a negatively charged lipid molecule. We have defined the potential binding site of the lipid molecule on KcsA by X-ray crystallographic analysis of a complex of KcsA with a monoclonal antibody Fab fragment. We also demonstrate that lipids are required for the in vitro refolding of the KcsA tetramer from the unfolded monomeric state. The correct refolding of the KcsA tetramer requires lipids, but it is not dependent on negatively charged lipids as refolding takes place in the absence of such lipids. We confirm that the presence of negatively charged lipids is required for ion conduction through the KcsA potassium channel, suggesting that the lipid bound to KcsA is important for ion channel function.

Amyloid-like features of polyglutamine aggregates and their assembly kinetics.

Abstract: The repeat length-dependent tendency of the polyglutamine sequences of certain proteins to form aggregates may underlie the cytotoxicity of these sequences in expanded CAG repeat diseases such as Huntington's disease. We report here a number of features of various polyglutamine (polyGln) aggregates and their assembly pathways that bear a resemblance to generally recognized defining features of amyloid fibrils. PolyGln aggregation kinetics displays concentration and length dependence and a lag phase that can be abbreviated by seeding. PolyGln aggregates exhibit classical beta-sheet-rich circular dichroism spectra consistent with an amyloid-like substructure. The fundamental structural unit of all the in vitro aggregates described here is a filament about 3 nm in width, resembling the protofibrillar intermediates in amyloid fibril assembly. We observed these filamentous structures either as isolated threads, as components of ribbonlike sheets, or, rarely, in amyloid-like twisted fibrils. All of the polyGln aggregates described here bind thioflavin T and shift its fluorescence spectrum. Although all polyGln aggregates tested bind the dye Congo red, only aggregates of a relatively long polyGln peptide exhibit Congo red birefringence, and this birefringence is only observed in a small portion of these aggregates. Remarkably, a monoclonal antibody with high selectivity for a generic amyloid fibril conformational epitope is capable of binding polyGln aggregates. Thus, polyGln aggregates exhibit most of the characteristic features of amyloid, but the twisted fibril structure with Congo red birefringence is not the predominant form in the polyGln repeat length range studied here. We also find that polyGln peptides exhibit an unusual freezing-dependent aggregation that appears to be caused by the freeze concentration of peptide and/or buffer components. This is of both fundamental and practical significance. PolyGln aggregation is revealed to be a highly specific process consistent with a significant degree of order in the molecular structure of the product. This ordered structure, or the assembly process leading to it, may be responsible for the cell-specific neuronal degeneration observed in Huntington's and other expanded CAG repeat diseases.

Dimers to doughnuts: redox-sensitive oligomerization of 2-cysteine peroxiredoxins.

Abstract: 2-Cys peroxiredoxins (Prxs) are a large and diverse family of peroxidases which, in addition to their antioxidant functions, regulate cell signaling pathways, apoptosis, and differentiation. These enzymes are obligate homodimers (alpha(2)), utilizing a unique intermolecular redox-active disulfide center for the reduction of peroxides, and are known to form two oligomeric states: individual alpha(2) dimers or doughnut-shaped (alpha(2))(5) decamers. Here we characterize both the oligomerization properties and crystal structure of a bacterial 2-Cys Prx, Salmonella typhimurium AhpC. Analytical ultracentrifugation and dynamic light scattering show that AhpC's oligomeric state is redox linked, with oxidization favoring the dimeric state. The 2.5 A resolution crystal structure (R = 18.5%, R(free) = 23.9%) of oxidized, decameric AhpC reveals a metastable oligomerization intermediate, allowing us to identify a loop that adopts distinct conformations associated with decameric and dimeric states, with disulfide bond formation favoring the latter. This molecular switch contains the peroxidatic cysteine and acts to buttress the oligomerization interface in the reduced, decameric enzyme. A structurally detailed catalytic cycle incorporating these ideas and linking activity to oligomeric state is presented. Finally, on the basis of sequence comparisons, we suggest that the enzymatic and signaling activities of all 2-Cys Prxs are regulated by a redox-sensitive dimer to decamer transition.

Heparin and other glycosaminoglycans stimulate the formation of amyloid fibrils from alpha-synuclein in vitro.

Abstract: Parkinson's disease is the second most common neurodegenerative disease and results from loss of dopaminergic neurons in the substantia nigra. The aggregation and fibrillation of alpha-synuclein have been implicated as a causative factor in the disease. Glycosaminoglycans (GAGs) are routinely found associated with amyloid deposits in most amyloidosis diseases, and there is evidence to support an active role of GAGs in amyloid fibril formation in some cases. In contrast to the extracellular amyloid deposits, the alpha-synuclein deposits in Lewy body diseases are intracellular, and thus it is less clear whether GAGs may be involved. To determine whether the presence of GAGs does affect the fibrillation of alpha-synuclein, the kinetics of fibril formation were investigated in the presence of a number of GAGs and other charged polymers. Certain GAGs (heparin, heparan sulfate) and other highly sulfated polymers (dextran sulfate) were found to significantly stimulate the formation of alpha-synuclein fibrils. Interestingly, the interaction of GAGs with alpha-synuclein is quite specific, since some GAGs, e.g., keratan sulfate, had negligible effect. Heparin not only increased the rate of fibrillation but also apparently increased the yield of fibrils. The molar ratio of heparin to alpha-synuclein and the incorporation of fluorescein-labeled heparin into the fibrils demonstrate that the heparin is integrated into the fibrils and is not just a catalyst for fibrillation. The apparent dissociation constant for heparin in stimulating alpha-synuclein fibrillation was 0.19 microM, indicating a strong affinity. Similar effects of heparin were observed with the A53T and A30P mutants of alpha-synuclein. Since there is some evidence that Lewy bodies may contain GAGs, these observations may be very relevant in the context of the etiology of Parkinson's disease.

Multiple Conformational Changes in Enzyme Catalysis

Abstract: Understanding the molecular mechanisms of enzyme catalysis and allosteric regulation has been a primary goal of biochemistry for many years. The dynamics of these processes, approached through a variety of kinetic methods, are discussed. The results obtained for many different enzymes suggest that multiple intermediates and conformations are general characteristics of the catalytic process and allosteric regulation. Ribonuclease, dihydrofolate reductase, chymotrypsin, aspartate aminotransferase, and aspartate transcarbamoylase are considered as specific examples. Typical and maximum rates of conformational changes and catalysis are also discussed, based on results obtained from model systems. The nature and rates of interconversion of the intermediates, along with structural information, can be used as the bases for understanding the incredible catalytic efficiency of enzymes. Potential roles of conformational changes in the catalytic process are discussed in terms of static and environmental effects, and in terms of dynamic coupling within the enzyme-substrate complex.

Kinetics and pathways of charge recombination in photosystem II.

Abstract: The mechanism of charge recombination of the S2QA- state in photosystem II was investigated by modifying the free energy gap between the quinone acceptor QA and the primary pheophytin acceptor Ph. This was done either by changing the midpoint potential of Ph (using mutants of the cyanobacterium Synechocystis with a modified hydrogen bond to this cofactor), or that of QA (using different inhibitors of the QB pocket). The results show that the recombination rate is dependent on the free energy gap between Ph and QA, which confirms that the indirect recombination pathway involving formation of Ph- has a significant contribution. In the mutant with the largest free energy gap, direct electron transfer from QA- to P+ predominates. The temperature dependence of the recombination rate was investigated, showing a lower activation enthalpy in this mutant compared with the WT. The data allow the determination of the rate of the direct route and of its relative weight in the various strains. The set of currently acc...

Islet amyloid: phase partitioning and secondary nucleation are central to the mechanism of fibrillogenesis.

Abstract: Islet amyloid polypeptide (IAPP) contributes to the pathogenesis of type II diabetes by depositing as cytotoxic amyloid fibers in the endocrine pancreas. Fiber formation occurs with a marked conformational change from an unstructured precursor. Using real-time quantitative kinetic methods, fibrillogenesis was characterized as a function of protein, denaturant, and seed concentration. Several observations are in sharp contrast to the expectations for nucleation-dependent polymerization. First, the half-time of conversion for both de novo and seeded kinetics were found to be independent of protein concentration. Second, while elongation kinetics scale linearly with protein concentration, they are relatively insensitive to changes in the total seed concentration. Third, seeded bypass of de novo fiber formation kinetics shows a lag phase. The seeded lag phase is eliminated by a time delay before the introduction of seed to a de novo reaction. Last, conversion is highly cooperative, with the time required for 10-90% conversion occurring much faster than the lag time. At a minimum, four kinetic steps are required to describe these observations: activation, fiber independent nucleation, fiber-dependent nucleation, and elongation. Furthermore, we invoke a phase transition in which protein initially forms an off-pathway dispersion. This single construct allows us to model both the concentration independence of the de novo reaction time and the first-order concentration dependence of the elongation kinetics. Marked acceleration of this reaction by hexafluoro-2-propanol reinforces this view by altering the relative solubility of the two phases and/or by stabilizing hydrogen-bonded structures in the transition states of the reaction pathway.

Toward a Unified Scheme for the Aggregation of Tau into Alzheimer Paired Helical Filaments

Abstract: Alzheimer's disease is characterized by aggregates of tau protein. Attempts to study the conditions for aggregation in vitro have led to different experimental systems, some of which appear mutually exclusive (e.g., oxidative vs reductive conditions, induction by polyanions vs fatty acids). We show here that different approaches and pathways can be viewed in a common framework, and that apparent differences can be explained by variations in the kinetics of subreactions. A unified view of PHF aggregation should help to analyze the causes of PHF aggregation and devise methods to prevent it.

Protofibrillar islet amyloid polypeptide permeabilizes synthetic vesicles by a pore-like mechanism that may be relevant to type II diabetes.

Abstract: Islet amyloid polypeptide (IAPP) and insulin are copackaged and cosecreted by pancreatic islet β-cells. Non-insulin-dependent (type II) diabetes mellitus (NIDDM) is characterized by dysfunction and...

Proteomic analysis of a highly active photosystem II preparation from the cyanobacterium Synechocystis sp. PCC 6803 reveals the presence of novel polypeptides.

Abstract: A highly active oxygen-evolving photosystem II (PSII) complex was purified from the HT-3 strain of the widely used cyanobacterium Synechocystis sp. PCC 6803, in which the CP47 polypeptide has been genetically engineered to contain a polyhistidine tag at its carboxyl terminus [Bricker, T. M., Morvant, J., Masri, N., Sutton, H. M., and Frankel, L. K. (1998) Biochim. Biophys. Acta 1409, 50-57]. These purified PSII centers had four manganese atoms, one calcium atom, and two cytochrome b(559) hemes each. Optical absorption and fluorescence emission spectroscopy as well as western immunoblot analysis demonstrated that the purified PSII preparation was devoid of any contamination with photosystem I and phycobiliproteins. A comprehensive proteomic analysis using a system designed to enhance resolution of low-molecular-weight polypeptides, followed by MALDI mass spectrometry and N-terminal amino acid sequencing, identified 31 distinct polypeptides in this PSII preparation. We propose a new nomenclature for the polypeptide components of PSII identified after PsbZ, which proceeds sequentially from Psb27. During this study, the polypeptides PsbJ, PsbM, PsbX, PsbY, PsbZ, Psb27, and Psb28 proteins were detected for the first time in a purified PSII complex from Synechocystis 6803. Five novel polypeptides were also identified in this preparation. They included the Sll1638 protein, which shares significant sequence similarity to PsbQ, a peripheral protein of PSII that was previously thought to be present only in chloroplasts. This work describes newly identified proteins in a highly purified cyanobacterial PSII preparation that is being widely used to investigate the structure, function, and biogenesis of this photosystem.

Cation−π Interactions in Ligand Recognition by Serotonergic (5-HT3A) and Nicotinic Acetylcholine Receptors: The Anomalous Binding Properties of Nicotine†

Abstract: A series of tryptophan analogues has been introduced into the binding site regions of two ion channels, the ligand-gated nicotinic acetylcholine and serotonin 5-HT3A receptors, using unnatural amino acid mutagenesis and heterologous expression in Xenopus oocytes. A cation−π interaction between serotonin and Trp183 of the serotonin channel 5-HT3AR is identified for the first time, precisely locating the ligand-binding site of this receptor. The energetic contribution of the observed cation−π interaction between a tryptophan and the primary ammonium ion of serotonin is estimated to be approximately 4 kcal/mol, while the comparable interaction with the quaternary ammonium of acetylcholine is approximately 2 kcal/mol. The binding mode of nicotine to the nicotinic receptor of mouse muscle is examined by the same technique and found to differ significantly from that of the natural agonist, acetylcholine.

Kinetic and Structural Studies on the Interaction of Cholinesterases with the Anti-Alzheimer Drug Rivastigmine

Abstract: Rivastigmine, a carbamate inhibitor of acetylcholinesterase, is already in use for treatment of Alzheimer's disease under the trade name of Exelon. Rivastigmine carbamylates Torpedo californica acetylcholinesterase very slowly (k(i) = 2.0 M(-1) min(-1)), whereas the bimolecular rate constant for inhibition of human acetylcholinesterase is >1600-fold higher (k(i) = 3300 M(-1) min(-1)). For human butyrylcholinesterase and for Drosophila melanogaster acetylcholinesterase, carbamylation is even more rapid (k(i) = 9 x 10(4) and 5 x 10(5) M(-1) min(-1), respectively). Spontaneous reactivation of all four conjugates is very slow, with <10% reactivation being observed for the Torpedo enzyme after 48 h. The crystal structure of the conjugate of rivastigmine with Torpedo acetylcholinesterase was determined to 2.2 A resolution. It revealed that the carbamyl moiety is covalently linked to the active-site serine, with the leaving group, (-)-S-3-[1-(dimethylamino)ethyl]phenol, being retained in the "anionic" site. A significant movement of the active-site histidine (H440) away from its normal hydrogen-bonded partner, E327, was observed, resulting in disruption of the catalytic triad. This movement may provide an explanation for the unusually slow kinetics of reactivation.

Hint, Fhit, and GalT: function, structure, evolution, and mechanism of three branches of the histidine triad superfamily of nucleotide hydrolases and transferases.

Abstract: HIT (histidine triad) proteins, named for a motif related to the sequence HφHφHφφ (φ, a hydrophobic amino acid), are a superfamily of nucleotide hydrolases and transferases, which act on the α-phosphate of ribonucleotides, and contain a ∼30 kDa domain that is typically either a homodimer of ∼15 kDa polypeptides with two active-sites or an internally, imperfectly repeated polypeptide that retains a single HIT active site. On the basis of sequence, substrate specificity, structure, evolution, and mechanism, HIT proteins can be classified into the Hint branch, which consists of adenosine 5‘-monophosphoramide hydrolases, the Fhit branch, which consists of diadenosine polyphosphate hydrolases, and the GalT branch, which consists of specific nucleoside monophosphate transferases, including galactose-1-phosphate uridylyltransferase, diadenosine tetraphosphate phosphorylase, and adenylyl sulfate:phosphate adenylytransferase. At least one human representative of each branch is lost in human diseases. Aprataxin, a ...