Characterization of Alzheimer's-like paired helical filaments from the core domain of tau protein using solid-state NMR spectroscopy

The polymerization of the microtubule-associated protein tau into paired helical filaments (PHFs) represents one of the hallmarks of Alzheimer's disease. We employed solid-state nuclear magnetic resonance (NMR) to investigate the structure and dynamics of PHFs formed in vitro by the three-repeat-domain (K19) of protein tau, representing the core of Alzheimer PHFs. While N and C termini of tau monomers in PHFs are highly dynamic and solvent-exposed, the rigid segment consists of three major beta-strands. Combination of through-bond and through-space ssNMR transfer methods with water-edited ((15)N, (13)C) and ((13)C, (13)C) correlation experiments suggests the existence of a fibril core that is largely built by repeat unit R3, flanked by surface-exposed units R1 and R4. Solid-state NMR, circular dichroism, and the fibrillization behavior of a K19 mutant furthermore indicate that electrostatic interactions play a central role in stabilizing the K19 PHFs.     

Spontaneous resolution through helical association of a Cu-azamacrocyclic complex with Lindqvist-type isopolyanion

Anionic metathesis reaction between the perchlorate salt of a copper-tetraazamacrocycle complex and the tetrabutylammonium salt of Lindqvist-type isopolyoxometalates in acetonitrile leads to the formation of two new inorganic-organic hybrid solids formulated as [Cu(L)(MeCN)][W(6)O(19)] (1) and [Cu(L)(MeCN)][Mo(6)O(19)] (2). Interestingly, both ion-pair complexes crystallize in a chiral space group P2(1)2(1)2(1). Crystallographic analysis of the obtained compounds reveals the occurrence of spontaneous resolution during crystallization. Both the enantiomorphs of compound 1 have been structurally characterized, whereas the resolution of compound 2 is rather poor.     

Dynamic unfolding of a regulatory subunit of cAMP-dependent protein kinase by capillary electrophoresis: Impact of cAMP dissociation on protein stability

Characterization of the unfolding dynamics of a recombinant type IA regulatory subunit (RIalpha) of cyclic adenosine monophosphate (cAMP)-dependent protein kinase (cAPK) was examined by CE with UV detection. Electrophoretic separation of RIalpha by CE in a buffer devoid of cAMP resulted in rapid dissociation of the complex from the original sample due to the high negative mobility of the ligand relative to receptor. This process enabled in-capillary generation of cAMP-stripped RIalpha, which was used to estimate the apparent dissociation constant (Kd) of 0.6 +/- 0.2 microM. A comparison of RIalpha dynamic unfolding processes with urea denaturation was performed by CE with (i.e., RIalpha-cAMP) and without (i.e., cAMP-stripped RIalpha) excess cAMP in the buffer during electromigration. The presence of cAMP in the buffer confirmed greater stabilization of the protein, as reflected by a higher standard free energy change (DeltaG(U) degrees) of 10.1 +/- 0.5 kcal x mol(+1) and greater cooperativity in unfolding (m) of -2.30 +/- 0.11 kcal x mol(-1) M(-1). CE offers a rapid, yet versatile platform for probing the thermodynamics of cAPK and other types of receptor-ligand complexes in free solution.     

Mapping of two networks of residues that exhibit structural and dynamical changes upon binding in a PDZ domain protein

We describe the changes in structure and dynamics that occur in the second PDZ domain of human tyrosine phosphatase 1E upon binding the small peptide RA-GEF2 by an analysis of NMR data based on their use as ensemble-averaged restraints in molecular dynamics simulations. This approach reveals the presence of two interconnected networks of residues, the first exhibiting structural changes and the second dynamical changes upon binding, and it provides a detailed mapping of the regions of increased and decreased mobility upon binding. Analysis of the dynamical properties of the residues in these networks reveals that conformational changes are transmitted through pathways of coupled side-chain reorientations. These results illustrate how the strategy we described, in which NMR data are used in combination with molecular dynamics simulations, can be used to characterize in detail the complex organization of the changes in structure and dynamics that take place in proteins upon binding.     

Copper-ion interaction with the 106-113 domain of the prion protein: a solution-equilibria study on model peptides

Prion diseases are characterized by a structural modification of the regular prion protein (PrP(C)) to its isoform, termed PrP(Sc)(scrapie). Such a modification involves the secondary and tertiary structure of the protein; the amino acidic sequence remains unchanged. PrP(Sc) is almost insoluble in non-denaturing solvents, resistant to proteases and it loses its redox activity. PrP(C) is able to bind copper and other metal ions: these complexes have been suggested to play an important role in the protein refolding leading to PrP(Sc). It is well-known that at least one relatively strong copper-binding site is located in the PrP(92--126) domain, where two His residues (96 and 111) are present. However, in the same domain, other amino acidic residues bear potentially donating atoms, i.e. Met, Asn and Lys residues. In order to shed light on the role of the side chains of such potentially tridentate amino acids on copper complexation, the polypeptide Ac-KTNMKHMA-NH(2), corresponding to the PrP(106--113) fragment, and some synthetic analogues have been investigated as ligands for the copper ion, by means of both thermodynamic and spectroscopic techniques. The pivotal role of imidazolic side chain of His in "anchoring" the metal ion has been confirmed. On the other hand, no clue was found on the participation of sulfur atom of Met or side amino-group of Lys residues to copper complex-formation.     

Structure determination of a peptide model of the repeated helical domain in Samia cynthia ricini silk fibroin before spinning by a combination of advanced solid-state NMR methods

Fibrous proteins unlike globular proteins, contain repetitive amino acid sequences, giving rise to very regular secondary protein structures. Silk fibroin from a wild silkworm, Samia cynthia ricini, consists of about 100 repeats of alternating polyalanine (poly-Ala) regions of 12-13 residues in length and Gly-rich regions. In this paper, the precise structure of the model peptide, GGAGGGYGGDGG(A)(12)GGAGDGYGAG, which is a typical repeated sequence of the silk fibroin, was determined using a combination of three kinds of solid-state NMR studies; a quantitative use of (13)C CP/MAS NMR chemical shift with conformation-dependent (13)C chemical shift contour plots, 2D spin diffusion (13)C solid-state NMR under off magic angle spinning and rotational echo double resonance. The structure of the model peptide corresponding to the silk fibroin structure before spinning was determined. The torsion angles of the central Ala residue, Ala(19), in the poly-Ala region were determined to be (phi, psi) = (-59 degrees, -48 degrees ) which are values typically associated with alpha-helical structures. However, the torsion angles of the Gly(25) residue adjacent to the C-terminal side of the poly-Ala chain were determined to be (phi, psi) = (-66 degrees, -22 degrees ) and those of Gly(12) and Ala(13) residues at the N-terminal of the poly-Ala chain to be (phi, psi) = (-70 degrees, -30 degrees ). In addition, REDOR experiments indicate that the torsion angles of the two C-terminal Ala residues, Ala(23) and Ala(24), are (phi, psi) = (-66 degrees, -22 degrees ) and those of N-terminal two Ala residues, Ala(13) and Ala(14) are (phi, psi) = (-70 degrees, -30 degrees ). Thus, the local structure of N-terminal and C-terminal residues, and also the neighboring residues of alpha-helical poly-Ala chain in the model peptide is a more strongly wound structure than found in typical alpha-helix structures.     

Molecular dynamics of cryptophane and its complexes with tetramethylammonium and neopentane using a continuum solvent model

Time scales currently obtainable in explicit–solvent molecular dynamics simulations are inadequate for the study of many biologically important processes. This has led to increased interest in the use of continuum solvent models. For such models to be used effectively, it is important that their behavior relative to explicit simulation be clearly understood. Accordingly, 5 ns stochastic dynamics simulations of a derivative of cryptophane-E alone, and complexed with tetramethylammonium and neopentane were carried out. Solvation electrostatics were accounted for via solutions to the Poisson equation. Nonelectrostatic aspects of solvation were incorporated using a surface area-dependent energy term. Comparison of the trajectories to those from previously reported 25 ns explicit–solvent simulations shows that use of a continuum solvent model results in enhanced sampling. Use of the continuum solvent model also results in a considerable increase in computational efficiency. The continuum solvent model is found to predict qualitative structural characteristics that are similar to those observed in explicit solvent. However, some differences are significant, and optimization of the continuum parameterization will be required for this method to become an efficient alternative to explicit–solvent simulation. ©1999 John Wiley & Sons, Inc. J Comput Chem 20: 956–970, 1999     

Light-controlled supramolecular helicity of a liquid crystalline phase using a helical polymer functionalized with a single chiroptical molecular switch

Control over the preferred helical sense of a poly(n-hexyl isocyanate) (PHIC) by using a single light-driven molecular motor, covalently attached at the polymer's terminus, has been accomplished in solution via a combination of photochemical and thermal isomerizations. Here, we report that after redesigning the photochromic unit to a chiroptical molecular switch, of which the two states are thermally stable but photochemically bistable, the chiral induction to the polymer's backbone is significantly improved and the handedness of the helical polymer is addressable by irradiation with two different wavelengths of light. Moreover, we show that the chiral information is transmitted, via the macromolecular level of the polyisocyanate, to the supramolecular level of a lyotropic cholesteric liquid crystalline phase consisting of these stiff, rodlike polymers. This allows the magnitude and sign of the supramolecular helical pitch of the liquid crystal film to be fully controlled by light.     

Examination of the folding of a short alanine-based helical peptide with salt bridges using molecular dynamics simulation

A molecular dynamics simulation of the folding of a short alanine-based helical peptide of 17 residues with three Glu...Lys (i, i + 4) salt bridge pairs, referred to as the AEK17 peptide, was carried out. The simulation gave an estimated simulation folding time of 2.5 ns, shorter than 12 ns for an alanine-based peptide of 16 residues with three Lys residues only, referred to as the AK16 peptide, simulated previously. After folded, the AEK17 peptide had a helical content of 77%, in excellent agreement with the experimentally determined value of 80%. An examination of the folding pathways of AEK17 indicated that the peptide proceeded via three-turn helix conformations more than the helix-turn-helix conformation in the folding pathways. An analysis of interactions indicated that the formation of hydrogen bonds between Lys residue side chains and backbone carbonyls is a major factor in the abundant conformation of the three-turn helix intermediate. The substitution of three Ala with Glu residues reduces the extent of hydrophobic interaction in alanine-based AK peptides with the result that the breaking of the interactions of Lys epsilon-NH3+(side chain)...C=O(backbone) is a major activation action for the AEK17 to achieve a complete fold, in contrast to the AK16 peptide, in which breaking non-native hydrophobic interaction is the rate-determining step.     

Quantitative estimation of stabilizers in petroleum and its compounds using a model reaction

The possible application of a kinetic model for the quantitative estimation of the inhibiting ability of natural compounds of unknown composition and structure has been studied. The antioxidative ability of several petroleums and their high-molecular weight components has been quantitatively estimated. Petroleums of different types possess an inhibiting activity, which is mainly determined by their tar components. In their antioxidative ability these substances equal and sometimes even surpass the activity of known commercial stabilizers.

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A molecular model of a point mutation (Val297Met) in the serine protease domain of protein C

A heterozygous GTG to ATG (Val297Met) mutation was detected in a patient with inherited protein C deficiency and deep vein thrombosis. Cosegregation of the mutation with protein C deficiency was observed through a family pedigree study. Molecular models of the serine protease domains of wild type and mutant protein C were constructed by standard comparative method. Val 297 was found to be located in the hydrophobic core of the protein. Although the substitution of Met for Val does not greatly alter the hydrophobicity of the protein, it introduces a bulkier side chain, which yields steric hindrance between this residue and adjacent residues, such as Met364, Tyr393, Ile321, Ile323, and Val378. It seems that the Met can not fit into the tight packing into which it is trapped, thereby probably inducing misfolding and/or greater instability of the protein. Such misfolding and/or instability thereby eventually disturbs the catalytic triad, in consistent with the observed type I deficiency state.     

Self-assembly of the beta2-microglobulin NHVTLSQ peptide using a coarse-grained protein model reveals a beta-barrel species

Although a wide variety of proteins can assemble into amyloid fibrils, the structure of the early oligomeric species on the aggregation pathways remains unknown at an atomic level of detail. In this paper we report, using molecular dynamics simulations with the OPEP coarse-grained force field, the free energy landscape of a tetramer and a heptamer of the beta2-microglobulin NHVTLSQ peptide. On the basis of a total of more than 17 ns trajectories started from various states, we find that both species are in equilibrium between amorphous and well-ordered aggregates with cross-beta-structure, a perpendicular bilayer beta-sheet, and, for the heptamer, six- or seven-stranded closed and open beta-barrels. Moreover, analysis of the heptamer trajectories shows that the perpendicular bilayer beta-sheet is one possible precursor of the beta-barrel, but that this barrel can also be formed from a twisted monolayer beta-sheet with successive addition of chains. Comparison with previous aggregation simulations and the fact that nature constructs transmembrane beta-sheet proteins with pores open the possibility that beta-barrels with small inner diameters may represent a common intermediate during the early steps of aggregation.     

Morphological evolution of inorganic crystal into zigzag and helical architectures with an exquisite association of polymer: a novel approach for morphological complexity

The morphology of potassium sulfate (K(2)SO(4)) crystals grown in a viscous polymer solution of poly(acrylic acid) (PAA) was remarkably changed from the tilted columnar assembly into zigzag and helical architectures with increasing PAA concentration. The habit modification of orthorhombic K(2)SO(4) with adsorption of PAA molecules on a specified crystal face fundamentally led to the formation of tilted unit crystals. Concurrently with the habit modification, a diffusion-limited condition controlling the assembly of tilted units was achieved in the presence of PAA molecules in the matrix. Various complex morphologies, including zigzag and helical assembly, emerged through the formation of twinned crystals with the variation of the diffusion condition. Understanding the morphogenesis observed in this report would provide a novel approach for sophisticated crystal design by using an exquisite association of organic and inorganic materials.     

Predictive in silico all-atom folding of a four-helix protein with a free-energy model

We report the predictive all-atom folding of the 60 amino acid four-helix bacterial ribosomal protein (BRP) L20 with a stochastic evolutionary optimization method in a free-energy force field. The energetically best, as well as six of the 10 lowest conformations, converge to a near-native structure. All of the 10 best energy conformations share the secondary structure elements of the native conformation, but differ in their tertiary alignment. The best conformation has a backbone root-mean-square deviation of 4.6 A to the native conformation and reproduces most distance constraints of the NMR experiment to 1.5 A resolution. Starting from random initial conditions, the native content of the simulated population increases more than 60-fold in the course of the simulation. These data demonstrate the feasibility of predictive unbiased all-atom protein folding with present day computational resources for the BRP L20.     

Isotachophoresis-based sample preparation of cellulases in sugarcane juice using bovine serum albumin as a model protein

The foremost requirement of quantification of cellulases expressed in genetically modified sugarcane is an efficient sample clean-up. This work investigates the feasibility of isotachophoresis for this purpose. An electrolyte system comprising a leading electrolyte of 10 mM formic acid at pH 9.0 and a terminating electrolyte of 10 mM β-alanine was devised and used to perform isotachophoresis of cellulases. The use of a simple front cutting method removed a majority of interfering species in the juice, thereby resulting in the formation of a distinct zone of desired proteins. In comparison to techniques such as ultrafiltration and liming, the analysis time and loss of desired proteins was lower when the sample was prepared by using isotachophoresis. Hence, isotachophoresis was an ideal choice for purification of the proteins in question from the remaining components in the juice.