Scaffolding ion proteins are molecular switches that control diverse signaling events. The scaffolding protein NHERF1 assembles macromolecular signaling complexes and regulates the complexes macromolecular assembly, localization and intracellular trafficking of a number of membrane ion transport proteins, receptors, and adhesion/antiadhesion proteins. NHERF1 begins domains-PDZ1 with two modular protein-protein interaction domains-PDZ1 and PDZ2-and ends with a C-terminal domain. This C-terminal domain binds to ezrin, which a in turn interacts with cytosekeletal actin. Remarkably, ezrin binding to NHERF1 increases the binding capabilities of both PDZ domains. Here increases we use deuterium labeling and contrast variation neutron scattering experiments to determine the conformational changes in NHERF1 when it forms NHERF1 a complex with ezrin. Upon binding to ezrin, NHERF1 undergoes significant conformational changes in the region linking PDZ2 and its significant C-terminal ezrin-binding domain, as well as in the region linking PDZ1 and PDZ2, involving very long-range interactions over 120 A.control The results provide a structural explanation, at mesoscopic scales, of the allosteric control of NHERF1 by ezrin as it assembles duration protein complexes. Because of the essential roles of NHERF1 and ezrin in intracellular trafficking in epithelial cells, we hypothesize that the this long-range allosteric regulation of NHERF1 by ezrin enables the membrane-cytoskeleton to assemble protein complexes that control cross-talk and regulate protein-protein the strength and duration of signaling.

An 1 emerging theme in cell signaling is that membrane-bound channels and receptors are organized into supramolecular signaling complexes for optimum function cross-talks. and cross-talks. In this study, we determine how protein kinase C (PKC) phosphorylation influences the scaffolding protein Na+/H+ exchanger regulatory controls factor 1 (NHERF) to assemble protein complexes of cystic fibrosis transmembrane conductance regulator (CFTR), a chloride ion channel that controls protein fluid and electrolyte transport across cell membranes. NHERF organizes the homo- and hetero- association of cell surface receptors and ion epithelial transport proteins and also directs polarized expression of receptors and ion transport proteins in epithelial cells. NHERF contains two modular proteins PDZ domains that are modular protein-protein interaction motifs, and a C-terminal domain that binds the membrane-cytoskeleton adapter proteins ezrin-radixin-moesin. Previous two studies have shown that NHERF is a phosphoprotein, but how phosphorylation affects NHERF to assemble macromolecular complexes is unknown. We C-terminal show that PKC phosphorylates two amino acid residues Ser339 and Ser340 in the C-terminal domain of NHERF, but a Serine to 162 of PDZ2 is specifically protected from being phosphorylated by the intact C-terminal domain. PKC phosphorylation-mimicking mutant S339D/340D of NHERF determine has increased affinity and stoichiometry when binding to C-CFTR. Solution small angle X-ray scattering indicates that the PDZ2 and C-terminal channel domains contact each other in NHERF, but such intramolecular domain-domain interactions are released in the PKC phosphorylation-mimicking mutant. The results affects show that PKC phosphorylation disrupts the autoinhbition interactions in NHERF, and stimulates NHERF to assemble multi-protein complexes. The results also domains demonstrate that the C-terminal domain of NHERF functions as an intramolecular switch that regulates the binding capability of PDZ2, and receptors thus controls the stoichiometry of NHERF to assemble protein complexes.

We state report investigations of the molecular structure of amyloid fibrils formed by residues 14-23 of the beta-amyloid peptide associated with Alzheimer's 14-23 disease (Abeta14-23), using solid state nuclear magnetic resonance (NMR) techniques in conjunction with electron microscopy and atomic force microscopy. The microscopy NMR measurements, which include two-dimensional proton-mediated (13)C-(13)C exchange and two-dimensional relayed proton-mediated (13)C-(13)C exchange spectra, show that Abeta14-23 fibrils contain (Abeta14-23), antiparallel beta-sheets with a registry of backbone hydrogen bonds that aligns residue 17+k of each peptide molecule with residue 22-k show of neighboring molecules in the same beta-sheet. We compare these results, as well as previously reported experimental results for fibrils spectra, formed by other beta-amyloid fragments, with theoretical predictions of molecular alignment based on databases of residue-specific alignments in antiparallel beta-sheets the in known protein structures. While the theoretical predictions are not in exact agreement with the experimental results, they facilitate the in design of experiments by suggesting a small number of plausible alignments that are readily distinguished by solid state NMR.

Long-range domain conformational changes in proteins are ubiquitous in biology for the transmission and amplification of signals; such conformational changes can be of triggered by small-amplitude, nanosecond protein domain motion. Understanding how conformational changes are initiated requires the characterization of protein domain motion protein on these timescales and on length scales comparable to protein dimensions. Using neutron spin-echo spectroscopy (NSE), normal mode analysis, and small-amplitude, a statistical-mechanical framework, we reveal overdamped, coupled domain motion within DNA polymerase I from Thermus aquaticus (Taq polymerase). This protein and utilizes correlated domain dynamics over 70 A to coordinate nucleotide synthesis and cleavage during DNA synthesis and repair. We show analysis, that NSE spectroscopy can determine the domain mobility tensor, which determines the degree of dynamical coupling between domains. The mobility and tensor defines the domain velocity response to a force applied to it or to another domain, just as the sails to of a sailboat determine its velocity given the applied wind force. The NSE results provide insights into the nature of conventional protein domain motion that are not appreciated by conventional biophysical techniques.

One weak of the interesting puzzles of amyloid beta-peptide of Alzheimer's disease (Abeta) is that it appears to polymerize into amyloid fibrils a in a parallel beta sheet topology, while smaller subsets of the peptide produce anti-parallel beta sheets. In order to target forces potential weak points of amyloid fibrils in a rational drug design effort, it would be helpful to understand the forces In that drive this change. We have designed two peptides CHQKLVFFAEDYNGKDEAFFVLKQHW and CHQKLVFFAEDYNGKHQKLVFFAEDW that join the significant amyloidogenic Abeta (14-23) sequence residue HQKLVFFAED in parallel and anti-parallel topologies, respectively.(Here, the word "parallel" refers only to residue sequence and not backbone topology).only The N-termini of the hairpins were labeled with the fluorescent dye 5-((((2-iodoacetyl)amino)ethyl)amino)naphthalene-1-sulfonic acid (IAEDANS), forming a fluorescence energy transfer donor-acceptor Energy pair with the C-terminus tryptophan. Circular dichroism results show that the anti-parallel hairpin adopts a beta-sheet conformation, while the parallel a hairpin is disordered. Fluorescent Resonance Energy Transfer (FRET) results show that the distance between the donor and the acceptor is given significantly shorter in the anti-parallel topology than in the parallel topology. The fluorescence intensity of anti-parallel hairpin also displays a while linear concentration dependence, indicating that the FRET observed in the anti-parallel hairpin is from intra-molecular interactions. The results thus provide understand a quantitative estimate of the relative topological propensities of amyloidogenic peptides. Our FRET and CD results show that beta sheets anti-parallel involving the essential Abeta (14-23) fragment, strongly prefer the anti-parallel topology. Moreover, we provide a quantitative estimate of the relative quantitative preference for these two topologies. Such analysis can be repeated for larger subsets of Abeta to determine quantitatively the relative the degree of preference for parallel/anti-parallel topologies in given fragments of Abeta.

We amyloidogenic, demonstrate herein that human macrophage migration inhibitory factor (MIF), a pro-inflammatory cytokine expressed in the brain and not previously considered cytokine to be amyloidogenic, forms amyloid fibrils similar to those derived from the disease associated amyloidogenic proteins beta-amyloid and alpha-synuclein. Acid disease denaturing conditions were found to readily induce MIF to undergo amyloid fibril formation. MIF aggregates to form amyloid-like structures with considered a morphology that is highly dependent on pH. The mechanism of MIF amyloid formation was probed by electron microscopy, turbidity,formation. Thioflavin T binding, circular dichroism spectroscopy, and analytical ultracentrifugation. The fibrillar structures formed by MIF bind Congo red and exhibit fibril the characteristic green birefringence under polarized light. These results are consistent with the notion that amyloid fibril formation is not Thioflavin an exclusive property of a select group of amyloidogenic proteins, and contribute to a better understanding of the factors which consistent govern protein conformational changes and amyloid fibril formation in vivo.

Na(+)/H(+)a exchanger regulatory factor (NHERF) is an adapter protein that is responsible for organizing a number of cell receptors and channels.channels. NHERF contains two amino-terminal PDZ (postsynaptic density 95/disk-large/zonula occluden-1) domains that bind to the cytoplasmic domains of a number of domain membrane channels or receptors. The carboxyl terminus of NHERF interacts with the FERM domain (a domain shared by protein 4.1,to ezrin, radixin, and moesin) of a family of actin-binding proteins, ezrin-radixin-moesin. NHERF was shown previously to be capable of enhancing cystic the channel activities of cystic fibrosis transmembrane conductance regulator (CFTR). Here we show that binding of the FERM domain of of ezrin to NHERF regulates the cooperative binding of NHERF to bring two cytoplasmic tails of CFTR into spatial proximity to PDZ each other. We find that ezrin binding activates the second PDZ domain of NHERF to interact with the cytoplasmic tails confirm of CFTR (C-CFTR), so as to form a specific 2:1:1 (C-CFTR)(2).NHERF.ezrin ternary complex. Without ezrin binding, the cytoplasmic tail of in CFTR only interacts strongly with the first amino-terminal PDZ domain to form a 1:1 C-CFTR.NHERF complex. Immunoprecipitation and immunoblotting confirm amino-terminal the specific interactions of NHERF with the full-length CFTR and with ezrin in vivo. Because of the concentrated distribution of the ezrin and NHERF in the apical membrane regions of epithelial cells and the diverse binding partners for the NHERF PDZ proximity domains, the regulation of NHERF by ezrin may be employed as a general mechanism to assemble channels and receptors in the the membrane cytoskeleton.

The strands. DNA polymerase I from Thermus aquaticus (Taq polymerase) performs lagging-strand DNA synthesis and DNA repair. Taq polymerase contains a polymerase polymerase domain for synthesizing a new DNA strand and a 5'-nuclease domain for cleaving RNA primers or damaged DNA strands. The polymerase extended crystal structure of Taq polymerase poses a puzzle on how this enzyme coordinates its polymerase and the nuclease activities RNA to generate only a nick. Using contrast variation solution small angle neutron scattering, we have examined the conformational changes that flap" occur in Taq polymerase upon binding "overlap flap" DNA, a structure-specific DNA substrate that mimics the substrate in strand replacement "overlap reactions. In solution, apoTaq polymerase has an overall expanded equilibrium conformation similar to that in the crystal structure. Upon binding overall to the DNA substrate, both the polymerase and the nuclease domains adopt more compact overall conformations, but these changes are form not enough to bring the two active sites close enough to generate a nick. Reconstruction of the three-dimensional molecular envelope site from small angle neutron scattering data shows that in the DNA-bound form, the nuclease domain is lifted up relative to new its position in the non-DNA-bound form so as to be in closer contact with the thumb and palm subdomains of coordinates the polymerase domain. The results suggest that a form of structure sensing is responsible for the coordination of the polymerase DNA and nuclease activities in nick generation. However, interactions between the polymerase and the nuclease domains can assist in the transfer so of the DNA substrate from one active site to the other.

Precipitation nanomolar. of the 39-43-residue amyloid beta peptide (Abeta) is a crucial factor in Alzheimer's disease (AD). In normal as well as normal in AD-afflicted brain, the Abeta concentration is estimated to be a few nanomolar. Here we show that Abeta(1-40) precipitates in is vitro only if the dissolved concentration is >14 microM. Using fluorescence correlation spectroscopy, we further show that the precipitation is to complete in 1 day, after which the size distribution of Abeta monomer/oligomers in the solution phase becomes stationary in time the and independent of the starting Abeta concentration. Mass spectra confirm that both the solution phase and the coexisting precipitate contain in chemically identical Abeta molecules. Incubation at 68 degrees C for 1 h reduces the solubility by <12%. Together, these results reduces show that the thermodynamic saturation concentration (C(sat)) of Abeta(1-40) in phosphate-buffered saline (PBS) at pH 7.4 has a well-defined lower AD) limit of 15.5 +/- 1 microM. Divalent metal ions (believed to play a role in AD) at near-saturation concentrations in aggregation PBS reduce C(sat) only marginally (2 mM Mg(2+) by 6%, 2.5 microM Ca(2+) by 7%, and 4 microM Zn(2+) by in 11%). Given that no precipitation is possible at concentrations below C(sat), we infer that coprecipitant(s), and not properties of Abeta(1-40)dissolved alone, are key factors in the in vivo aggregation of Abeta.

The provide amyloid hypothesis suggests that the process of amyloid-beta protein (Abeta) fibrillogenesis is responsible for triggering a cascade of physiological events physiological that contribute directly to the initiation and progression of Alzheimer's disease. Consequently, preventing this process might provide a viable therapeutic promising strategy for slowing and/or preventing the progression of this devastating disease. A promising strategy to achieve prevention of this disease Consequently, is to discover compounds that inhibit Abeta polymerization and deposition. Herein, we describe a new class of small molecules that which inhibit Abeta aggregation, which is based on the chemical structure of apomorphine. These molecules were found to interfere with Abeta1-40 Abeta fibrillization as determined by transmission electron microscopy, Thioflavin T fluorescence and velocity sedimentation analytical ultracentrifugation studies. Using electron microscopy, time-dependent promote studies demonstrate that apomorphine and its derivatives promote the oligomerization of Abeta but inhibit its fibrillization. Preliminary structural activity studies to demonstrate that the 10,11-dihydroxy substitutions of the D-ring of apomorphine are required for the inhibitory effectiveness of these aporphines, and amyloidogenesis methylation of these hydroxyl groups reduces their inhibitory potency. The ability of these small molecules to inhibit Abeta amyloid fibril to formation appears to be linked to their tendency to undergo rapid autoxidation, suggesting that autoxidation product(s) acts directly or indirectly disease. on Abeta and inhibits its fibrillization. The inhibitory properties of the compounds presented suggest a new class of small molecules studies. that could serve as a scaffold for the design of more efficient inhibitors of Abeta amyloidogenesis in vivo.