Synthesis and ITC characterization of novel nanoparticles constituted by poly(gamma-benzyl L-glutamate)-beta-cyclodextrin

Imparting desired technological characteristics to polymeric nanoparticles requires the development of original polymers. In the present work, the synthesis and characterization of a novel PBLG-derivative, the poly(gamma-benzyl L-glutamate)-beta-cyclodextrin (PBLG-beta-CD-50), have been carried out. Nanoparticles from either PBLG-beta-CD-50 polymer or from mixtures with PBLG have been prepared using a modified nanoprecipitation method. Spherically shaped nanoparticles with diameter in the range of 50-70 nm were obtained, as determined by dynamic laser light scattering and transmission electron microscopy. The presence of a surfactant in the suspension medium had almost no influence on these parameters and was not necessary to the shelf-stability of the suspension. Further, isothermal titration microcalorimetry (ITC) experiments have been used to show unambiguously that about 20% of the cyclodextrins remain functional within the particles. Consequently, this system may be of interest when association of large amounts of hydrophobic drugs to nanoparticles is required.     

Geometrical constraints limiting the poly(ADP‐ribose) conformation investigated by molecular dynamics simulation

Poly(ADP‐ribosylation) is a post‐transductional modification that regulates protein's function. Most of the proteins subjected to this control mechanism belong to machineries involved in DNA damage repair, or DNA interacting proteins. Poly(ADP‐ribose) polymers are long chains of even 100 monomer length that can be branched at several positions but, not withstanding its importance, nothing is known concerning its structure. To understand, which are the geometrical parameters that confer to the polymer the structural constraints that determine its interaction with the target proteins, we have performed molecular dynamics of three chains of different length, made by 5, 25, and 30 units, the last one being branched. Analysis of the simulations allowed us to identify the main intra‐ and inter‐monomer dihedral angles that govern the structure of the polymer that however, does not reach a unique definite conformation. © 2013 Wiley Periodicals, Inc. Biopolymers 101: 78–86, 2014.     

Structure and hydration of the amylopectin trisaccharide building blocks--Synthesis, NMR, and molecular dynamics

To gain insight into the molecular details and hydration of amylopectin, the five constituting trisaccharides have been chemically synthesized as their methyl alpha-glycosides. All five trisaccharides were subjected to 950 MHz NMR spectroscopy for complete assignment and nanosecond molecular dynamics trajectories were calculated to study the structure and dynamics of the trisaccharides in aqueous solution. Systematic analysis of the simulation data revealed several examples of bridging water molecules playing an important role in the stabilization of specific amylopectin conformations, which was also supported by the experimental NMR data such as interresidue NOE's and heteronuclear scalar couplings between nuclei from neighboring residues. Although alpha-maltotriose, alpha-iso-maltotriose, alpha-panose and alpha-isopanose are relatively well characterized structures, the study also includes one less characterized trisaccharide with the structure alphaGlcp(1-->4)alphaGlcp(1-->6)alphaGlcp. This trisaccharide, tentatively labelled alpha-forkose, is located at the branch point of amylopectin, forking the amylopectin into two strands that align into double-helical segments. The results show that the conformation of alpha-forkose takes a natural bend form which fits well into the structure of the double-helical segment of amylopectin. As the only trisaccharide in this study the structure of alpha-forkose is not significantly influenced by the hydration. In contrast, alpha-isopanose takes a restricted, but rather extended form due to an exceptionally strong localized water density. The two homo-linkage oligomers, alpha-maltotriose and alpha-iso-maltotriose, showed to be the most extended and the most flexible trimers, respectively, providing regular structure for crystalline domains and maximum linker flexibility for amorphous domains.     

On the flexibility of poly(γ-benzyl L-glutamate) in helicogenic solvents

The molecular-weight dependence of the rms radius of gyration of poly(γ-benzyl L -glutamate) (PBLG) in helicogenic solvents shows negative and positive deviations from expectations for an intact and rigid α-helix in the higher and lower molecular-weight ranges, respectively. In order to study the reason for both deviations, we compare the extant experimental data of with those computed for wormlike chain, freely jointed rod, and a rigid rod having random-coil portions at both ends. The computation for the freely jointed rod and the rigid rod having frayed ends is carried out by a simulation method of Muroga. From the Zimm and Bragg theory and the above comparisons, it is concluded that both deviations can be self-consistently explained if PBLG in helicogenic solvents has an essentially intact α-helical structure with some flexibility arising from random fluctuations in hydrogen bond length. This flexibility explains the negative deviations in the high molecular weight region. The positive deviations in the low molecular weight region result from the tendency of helices to unwind at the ends. © 1998 John Wiley & Sons, Inc. Biopoly 45: 281–288, 1998     

Synthesis and secondary structure of oligo(alpha-isobutyl beta,L-aspartate)s

The oligo(beta-peptide)s, hexa(alpha-isobutyl beta,L-aspartate) (Hex-AIBLA) and octa(alpha-isobutyl beta,L-aspartate) (Oct-AIBLA), were synthesized in solution by using standard coupling methods. Powder x-ray diffraction showed that the octamer crystallized in the two helical crystal forms known to exist in the homologous poly(beta-peptide), whereas the hexamer seemed to adopt an extended conformation. Both CD and 1H-NMR spectra of Oct-AIBLA in MeOH revealed the presence of a regularly folded conformation in this solvent, presumably the 3(14) helix. The helix-to-coil transition of Oct-AIBLA was observed to take place upon heating in both MeOH and CHCl3, in the second case associated with a not-well-defined aggregation-disaggregation process. The spectroscopic evidence obtained on the presence of folded structures in Hex-AIBLA were much weaker than for the octamer.     

A molecular dynamics study of the 41-56 beta-hairpin from B1 domain of protein G.

The structural and dynamical behavior of the 41-56 beta-hairpin from the protein G B1 domain (GB1) has been studied at different temperatures using molecular dynamics (MD) simulations in an aqueous environment. The purpose of these simulations is to establish the stability of this hairpin in view of its possible role as a nucleation site for protein folding. The conformation of the peptide in the crystallographic structure of the protein GB1 (native conformation) was lost in all simulations. The new equilibrium conformations are stable for several nanoseconds at 300K (>10 ns), 350 K (>6.5 ns), and even at 450 K (up to 2.5 ns). The new structures have very similar hairpin-like conformations with properties in agreement with available experimental nuclear Overhauser effect (NOE) data. The stability of the structure in the hydrophobic core region during the simulations is consistent with the experimental data and provides further evidence for the role played by hydrophobic interactions in hairpin structures. Essential dynamics analysis shows that the dynamics of the peptide at different temperatures spans basically the same essential subspace. The main equilibrium motions in this subspace involve large fluctuations of the residues in the turn and ends regions. Of the six interchain hydrogen bonds, the inner four remain stable during the simulations. The space spanned by the first two eigenvectors, as sampled at 450 K, includes almost all of the 47 different hairpin structures found in the database. Finally, analysis of the hydration of the 300 K average conformations shows that the hydration sites observed in the native conformation are still well hydrated in the equilibrium MD ensemble.     

C(alpha)-hydroxymethyl methionine: synthesis, optical resolution and crystal structure of its (+)-N(alpha)-benzoyl derivative.

(R, S)-Methionine was transformed into C(alpha)-hydroxymethyl methionine by a route involving C(alpha)-hydroxymethylation of 2-phenyl-4-methylthioethyl-5-oxo-4,5-dihydro-1,3-oxazole. The absolute configuration of (-)-C(alpha)-hydroxymethyl methionine was elucidated to be (S) by chemical correlation with (S) (-)-C(alpha)-ethyl serine. Absolute structure determination (by single crystal X-ray diffraction) on N(alpha)-benzoyl-C(alpha)-hydroxymethyl methionine confirmed the (R)-configuration for the (+)-enantiomer. In addition, the X-ray diffraction analysis showed that the C(alpha,alpha)-disubstituted glycyl residue adopts the fully extended (C5) conformation.     

Conformation of poly(γ‐glutamic acid) in aqueous solution

Local conformation and overall conformation of poly(γ‐DL‐glutamic acid) (PγDLGA) and poly(γ‐L‐glutamic acid) (PγLGA) in aqueous solution was studied as a function of degree of ionization ε by ¹H‐NMR, circular dichroism, and potentiometric titration. It was clarified that their local conformation is represented by random coil over an entire ε range and their overall conformation is represented by expanded random‐coil in a range of ε > ε^*, where ε^* is about 0.3, 0.35, 0.45, and 0.5 for added‐salt concentration of 0.02M, 0.05M, 0.1M, and 0.2M, respectively. In a range of ε < ε^*, however, ε dependence of their overall conformation is significantly differentiated from each other. PγDLGA tends to aggregate intramolecularly and/or intermolecularly with decreasing ε, but PγLGA still behaves as expanded random‐coil. It is speculated that spatial arrangement of adjacent carboxyl groups along the backbone chain essentially affects the overall conformation of PγGA in acidic media. © 2015 Wiley Periodicals, Inc. Biopolymers 105: 191–198, 2016.     

Conformational investigation of alpha,beta-dehydropeptides. XVI. Beta-turn tendency in Ac-Pro-DeltaXaa-NHMe: crystallographic and theoretical studies.

The crystal structures of two diastereomeric alpha,beta-dehydrobutyrine peptides Ac-Pro-(Z)-DeltaAbu-NHMe (I) and Ac-Pro-(E)-DeltaAbu-NHMe (II) have been determined. Both dehydropeptides adopt betaI-turn conformation characterized by the pairs of (phi(i+1), psi(i+1)) and (phi(i+2), psi(i+2)) angles as -66, -19, -97, 11 degrees for I and -59, -27, -119, 29 degrees for II. In each peptide, the betaI turn is stabilized by (i + 3) --> i intramolecular hydrogen bonds with N...O distance of 3.12 A for I and 2.93 A for II. These structures have been compared to the crystal structures of homologous peptides Ac-Pro-DeltaVal-NHMe and Ac-Pro-DeltaAla-NHMe. Theoretical analyses by DFT/B3LYP/6-31 + G** method of conformers formed by these four peptides and by the saturated peptide Ac-Pro-Ala-NHMe revealed that peptides with a (Z) substituent at the C(beta) (i+2) atom of dehydroamino acid, i.e. Ac-Pro-DeltaVal-NHMe and Ac-Pro-(Z)-DeltaAbu-NHMe, predominantly form beta turns, both in vacuo and in polar environment. The tendency to adopt beta-turn conformation is much weaker for the peptides lacking the (Z) substituent, Ac-Pro-(E)-DeltaAbu-NHMe and Ac-Pro-DeltaAla-NHMe. The latter adopts a semi-extended or an extended conformation in every polar environment, including a weakly polar solvent. The saturated peptide Ac-Pro-Ala-NHMe in vacuo prefers a beta-turn conformation, but in polar environment the differences between various conformers are small. The role of pi-electron correlation and intramolecular hydrogen bonds interaction in stabilizing the hairpin structures are discussed.     

Formation of oriented polypeptides on Au(111) surface depends on the secondary structure controlled by peptide length.

We synthesized three different lengths of poly(L-lysine) containing an -SH group at the terminal (PLL(n)-SH, n (polymerization degree) = 4, 10, 30) and adsorbed them on an Au(111) surface. To analyze the formation process and the structure of self-assembled monolayers (SAMs), we used atomic force microscopy (AFM) and Fourier transform infrared reflection absorption spectra (FT-IR RAS). At the initial stage of SAM growth, formation of nanosize domains was confirmed by AFM imaging. The alpha-helical PLL(30)-SH exhibited a well-defined SAM structure after adsorption reached equilibrium. The alpha-helical PLL(30)-SH was almost perpendicular to the gold surface and exhibited interesting molecular packing due to the secondary structure of PLL(30)-SH and the underlying Au(111) array. The tilt angle of the helix axis from the substrate normal was estimated to be about 50 degrees (AFM) and 44 degrees (FT-IR RAS) respectively. On the other hand, PLL(4)-SH and PLL(10)-SH formed beta-sheet-type SAMs on the Au(111) surface based on the structure determined by FT-IR RAS spectrum.     

Determination of alpha-helix and beta-sheet stability in the solid state: a solid-state NMR investigation of poly(L-alanine)

The relative stability of alpha-helix and beta-sheet secondary structure in the solid state was investigated using poly(L-alanine) (PLA) as a model system. Protein folding and stability has been well studied in solution, but little is known about solid-state environments, such as the core of a folded protein, where peptide packing interactions are the dominant factor in determining structural stability. (13)C cross-polarization with magic angle spinning (CPMAS) NMR spectroscopy was used to determine the backbone conformation of solid powder samples of 15-kDa and 21.4-kDa PLA before and after various sample treatments. Reprecipitation from helix-inducing solvents traps the alpha-helical conformation of PLA, although the method of reprecipitation also affects the conformational distribution. Grinding converts the secondary structure of PLA to a final steady-state mixture of 55% beta-sheet and 45% alpha-helix at room temperature regardless of the initial secondary structure. Grinding PLA at liquid nitrogen temperatures leads to a similar steady-state mixture with 60% beta-sheet and 40% alpha-helix, indicating that mechanical shear force is sufficient to induce secondary structure interconversion. Cooling the sample in liquid nitrogen or subjecting it to high pressure has no effect on secondary structure. Heating the sample without grinding results in equilibration of secondary structure to 50% alpha-helix/50% beta-sheet at 100 degrees C when starting from a mostly alpha-helical state. No change was observed upon heating a beta-sheet sample, perhaps due to kinetic effects and the different heating rate used in the experiments. These results are consistent with beta-sheet approximately 260 J/mol more stable than alpha-helix in solid-state PLA.     

Thermodynamic and kinetic characterization of a beta-hairpin peptide in solution: an extended phase space sampling by molecular dynamics simulations in explicit water

The folding of the amyloidogenic H1 peptide MKHMAGAAAAGAVV taken from the syrian hamster prion protein is explored in explicit aqueous solution at 300 K using long time scale all-atom molecular dynamics simulations for a total simulation time of 1.1 mus. The system, initially modeled as an alpha-helix, preferentially adopts a beta-hairpin structure and several unfolding/refolding events are observed, yielding a very short average beta-hairpin folding time of approximately 200 ns. The long time scale accessed by our simulations and the reversibility of the folding allow to properly explore the configurational space of the peptide in solution. The free energy profile, as a function of the principal components (essential eigenvectors) of motion, describing the main conformational transitions, shows the characteristic features of a funneled landscape, with a downhill surface toward the beta-hairpin folded basin. However, the analysis of the peptide thermodynamic stability, reveals that the beta-hairpin in solution is rather unstable. These results are in good agreement with several experimental evidences, according to which the isolated H1 peptide adopts very rapidly in water beta-sheet structure, leading to amyloid fibril precipitates [Nguyen et al., Biochemistry 1995;34:4186-4192; Inouye et al., J Struct Biol 1998;122:247-255]. Moreover, in this article we also characterize the diffusion behavior in conformational space, investigating its relations with folding/unfolding conditions.     

A study of conformational stability of poly(L-alanine), poly(L-valine), and poly(L-alanine)/poly(L-valine) blends in the solid state by (13)C cross-polarization/magic angle spinning NMR

13C cross-polarization/magic angle spinning (CP/MAS) NMR and (1)H T(1rho) experiments of poly(L-alanine) (PLA), poly(L-valine) (PLV), and PLA/PLV blends have been carried out in order to elucidate the conformational stability of the polypeptides in the solid state. These were prepared by adding a trifluoroacetic acid (TFA) solution of the polymer with a 2.0 wt/wt % of sulfuric acid (H(2)SO(4)) to alkaline water. From these experimental results, it is clarified that the conformations of PLA and PLV in their blends are strongly influenced by intermolecular hydrogen-bonding interactions that cause their miscibility at the molecular level.     

Relationship between conformation and geometry as evidenced by molecular dynamics simulation of Cα,α-dialkylated glycines

The relationship between the local backbone conformation and bond angles at C^α of symmetrically substituted C^α,α-dialkylated glycines (C^α,α-dimethylglycine or α-aminoisobutyric acid, Aib; C^α,α-diethylglycine, Deg; C^α,α-di-n-propylglycine, Dpg) has been investigated by molecular dynamics (MD) simulation adopting flat bottom harmonic potentials, instead of the usual harmonic restraints, for the C^α bond angles. The MD simulations show that the C^α bond angles are related to the local backbone conformation, irrespectively of the side-chain length of Aib, Deg, and Dpg residues. Moreover, the N-C^α-C′ (τ) angle is the most sensitive conformational parameter and, in the folded form, is always larger and more flexible than in the extended one. © 1998 John Wiley & Sons, Inc. Biopoly 46: 239–244, 1998     

Folding and stability of the three-stranded beta-sheet peptide Betanova: insights from molecular dynamics simulations

The dynamics of the three-stranded beta-sheet peptide Betanova has been studied at four different temperatures (280, 300, 350, and 450 K by molecular dynamics simulation techniques, in explicit water. Two 20-ns simulations at 280 K indicate that the peptide remains very flexible under "folding" conditions sampling a range of conformations that together satisfy the nuclear magnetic resonance (NMR)-derived experimental constraints. Two simulations at 300 K (above the experimental folding temperature) of 20 ns each show partial formation of "native"-like structure, which also satisfies most of the NOE constraints at 280 K. At higher temperature, the presence of compact states, in which a series of hydrophobic contacts remain present, are observed. This is consistent with experimental observations regarding the role of hydrophobic contacts in determining the peptide's stability and in initiating the formation of turns and loops. A set of different structures is shown to satisfy NMR-derived distance restraints and a possible mechanism for the folding of the peptide into the NMR-determined structure is proposed.     

A quantitative anharmonic analysis of the amide A band in α‐helical poly(L‐alanine)

Polarized ir spectra of oriented films of α‐helical poly(l ‐alanine) (α‐PLA) have been obtained as a function of residual solvent dichloroacetic acid (DCA). The amide A, B, II, and V regions exhibit multiple bands whose structure depends on the residual DCA content, and those associated with the α_I‐PLA structure have been identified. A calculation of the relevant cubic anharmonic force constants indicates that, contrary to previous assignments, the overtone of amide II(A) is in Fermi resonance with the NH stretch fundamental, whose unperturbed frequency we now find to be at 3314 cm⁻¹, significantly higher than the previously suggested 3279 cm⁻¹. The presence of a structure in addition to the standard α_I‐PLA is indicated by our analysis. © 1999 John Wiley & Sons, Inc. Biopoly 49: 195–207, 1999     

A detailed unfolding pathway of a (beta/alpha)8-barrel protein as studied by molecular dynamics simulations

The (beta/alpha)(8)-barrel is the most common protein fold. Similar structural properties for folding intermediates of (beta/alpha)(8)-barrel proteins involved in tryptophan biosynthesis have been reported in a number of experimental studies; these intermediates have the last two beta-strands and three alpha-helices partially unfolded, with other regions of the polypeptide chain native-like in conformation. To investigate the detailed folding/unfolding pathways of these (beta/alpha)(8)-barrel proteins, temperature-induced unfolding simulations of N-(5'-phosphoribosyl)anthranilate isomerase from Escherichia coli were carried out using a special-purpose parallel computer system. Unfolding simulations at five different temperatures showed a sequential unfolding pathway comprised of several events. Early events in unfolding involved disruption of the last two strands and three helices, producing an intermediate ensemble similar to those detected in experimental studies. Then, denaturation of the first two betaalpha units and separation of the sixth strand from the fifth took place independently. The remaining central betaalphabetaalphabeta module persisted the longest during all simulations, suggesting an important role for this module as the incipient folding scaffold. Our simulations also predicted the presence of a nucleation site, onto which several hydrophobic residues condensed forming the foundation for the central betaalphabetaalphabeta module.     

Immunohistochemical detection of the retinoid acid receptors (RXR-alpha, -beta, -gamma) and Farnesoid X-activated receptor (FXR) in the marbled newt along the annual cycle

Retinoid acid receptors (RXR-alpha, -beta, -gamma) and Farnesoid X-activated receptor (FXR) expression in the testis of the marbled newt were investigated with special attention to the changes during the annual testicular cycle, using light microscopy immunohistochemistry and Western blot analysis. The annual testicular cycle of the marbled newt (Triturus marmoratus marmoratus) comprises three periods: (a) proliferative period (germ cell proliferation from primordial germ cells to round spermatids, April-June); (b) spermiogenesis period (July-September); and (c) quiescence period (interstitial and follicular cells form the glandular tissue, October-April). In the proliferative period, primordial germ cells and primary spermatogonia immunostained intensely to the three types of RXRs and also to FXR. In the other periods, immunostaining to these antibodies was weak or absent. Secondary spermatogonia stained weakly to the four antibodies in the proliferative period, and only to FXR, also weakly, in the spermiogenesis period. Immunoreactive primary spermatocytes were weakly labeled with the RXR antibodies in the proliferative period. Spermatids and spermatozoa did not stain to any antibody in any period. Follicular cells only immunostained to RXR-gamma and only in the quiescence period when they are forming the glandular tissue, together with the interstitial cells. As follicular cells, interstitial cells only immunostained in the quiescence period; however, they immunoreacted to the three types of RXRs. These findings suggest that in the newt, RXRs and FXR are involved in spermatogenesis control by regulating the proliferation of primordial germ cells and spermatogonia. In addition, RXR-gamma seems to be also involved in the development of the glandular (steroidogenic) tissue.     

Facile transition between 3(10)- and alpha-helix: structures of 8-, 9-, and 10-residue peptides containing the -(Leu-Aib-Ala)2-Phe-Aib- fragment.

A structural transition from a 3(10)-helix to an alpha-helix has been characterized at high resolution for an octapeptide segment located in 3 different sequences. Three synthetic peptides, decapeptide (A) Boc-Aib-Trp-(Leu-Aib-Ala)2-Phe-Aib-OMe, nonapeptide (B) Boc-Trp-(Leu-Aib-Ala)2-Phe-Aib-OMe, and octapeptide (C) Boc-(Leu-Aib-Ala)2-Phe-Aib-OMe, are completely helical in their respective crystals. At 0.9 A resolution, R factors for A, B, and C are 8.3%, 5.4%, and 7.3%, respectively. The octapeptide and nonapeptide form ideal 3(10)-helices with average torsional angles phi(N-C alpha) and psi(C alpha-C') of -57 degrees, -26 degrees C and -60 degrees, -27 degrees for B. The 10-residue peptide (A) begins as a 3(10)-helix and abruptly changes to an alpha-helix at carbonyl O(3), which is the acceptor for both a 4-->1 hydrogen bond with N(6)H and a 5-->1 hydrogen with N(7)H, even though the last 8 residues have the same sequence in all 3 peptides. The average phi, psi angles in the decapeptide are -58 degrees, -28 degrees for residues 1-3 and -63 degrees, -41 degrees for residues 4-10. The packing of helices in the crystals does not provide any obvious reason for the transition in helix type. Fourier transform infrared studies in the solid state also provide evidence for a 3(10)- to alpha-helix transition with the amide I band appearing at 1,656-1,657 cm-1 in the 9- and 10-residue peptides, whereas in shorter sequences the band is observed at 1,667 cm-1.     

1alpha,25-Dihydroxyvitamin D(3) inhibits rat liver ultrastructural changes and the development of gamma-glutamyltranspeptidase-positive foci in diethylnitrosamine-initiated and streptozotocin-induced diabetes-promoted hepatocarcinogenesis

In the present study, the chemopreventive effect of the active metabolite of vitamin D, 1alpha,25-dihydroxyvitamin D(3) (VD(3)), against chemically-induced and diabetes-promoted rat liver carcinogenesis was investigated. Hepatocarcinogenesis was initiated with a single intraperitoneal (i.p.) injection of diethylnitrosamine (DEN) (125 mg kg(-1) body weight) at week 4 followed by promotion with streptozotocin (STZ) (65 mg kg(-1) body weight with a single i.p. injection) at week 7. With this basic experimental regimen, the effect of VD(3) (0.3 microg (0.1 ml)(-1) propylene glycol per os twice a week) was investigated with effect from 4 weeks prior to the exposure of DEN. The results showed that VD(3) supplementation throughout the experimental period reduced the incidence, total number and multiplicity and altered the size of visible persistent nodules (PNs) in DEN- or DEN + STZ-treated rats as compared with their respective controls. In these two groups, it also caused a significant decrease in the number (p < 0.002 and 0.001 respectively) and focal area (p < 0.05) of gamma-glutamyltranspeptidase (GGT)-positive hepatic foci. Moreover, continuous supplementation of VD(3) exhibits a protective effect in maintaining the normal cellular architecture of the hepatocytes in DEN- or DEN + STZ-treated rats. Our results thus strongly suggest that VD(3) is very effective in the inhibition of DEN-initiated and STZ-induced diabetes-promoted rat liver carcinogenesis.