Effect of side-chain length on the side-chain dynamics of alpha-helical poly(L-glutamic acid) as probed by a fluorescence blob model

Two series of pyrene-labeled poly(glutamic acid) (Py-PGA) were synthesized utilizing two different linkers for pyrene attachment, namely 1-pyrenemethylamine (PMA) and 1-pyrenebutylamine (PBA). Several Py-PGAs were synthesized for each series with pyrene contents ranging from 4 to 15 mol %. Py-PGA forms a rigid alpha-helix in DMF that effectively locks the backbone in place, thus enabling only side-chain or linker motions to be monitored by time-resolved fluorescence. Time-resolved fluorescence decays were acquired for the pyrene monomer of the Py-PGA constructs and the fluorescence blob model (FBM) was used to quantify the dynamics of the different linkers connecting pyrene to the backbone. Nitromethane was used to shorten the lifetime of the pyrene monomer, in effect controlling the probing time of the pyrene group, from 50 to 155 ns for PGA-PBA and from 50 to 215 ns for PGA-PMA. The FBM analysis of the fluorescence decays led to the conclusion that excimer formation around the rigid alpha-helix backbone takes place in a compact environment. The number of glutamic acid units within a blob, N blob, decreased only slightly with decreasing probing time for both Py-PGA constructs as a result of the compact distribution of the chromophores around the alpha-helix. The PGA alpha-helix was modeled using Hyperchem software and the ability of two pyrene groups to encounter was evaluated as they were separated by increasing numbers of amino acids along the alpha-helix. The number of amino acids required for two pyrenes to lose their ability to overlap and form excimer matched closely the N blob values retrieved using the FBM.     

Study of the semidilute solutions of poly(N,N-dimethylacrylamide) by fluorescence and its implications to the kinetics of coil-to-globule transitions

The large scale motions of poly(N,N-dimethylacrylamide) chains randomly labeled with pyrene (Py-PDMA) were monitored by steady-state and time-resolved fluorescence in semidilute solutions of naked PDMA in acetone and DMF for polymer concentrations ranging from 0 to 550 g/L. Although increasing the polymer concentration of the solution led to a decrease of the mobility of the chromophore attached onto the PDMA backbone, this reduction was rather modest when compared to the large increase of the macroscopic viscosity. This result indicated that locally, the monomer constituting the chains experienced freedom of movement despite the high solution viscosity. The restricted mobility of the chromophore was characterized by the number of monomers occupying the volume probed by the excited chromophore during its lifetime, referred to as a fluorescence "blob". The number of monomers constituting a fluorescence blob, N(F)(-)(blob), and the volume of a fluorescence blob, V(F)(-)(blob), were found to decrease as the polymer concentration of the solution increased, reflecting the decreased mobility experienced by the chromophore. In DMF, the radius of an F-blob was found to scale as N(nu)(F)(-)blob, where nu equaled 0.66 +/- 0.03, very close to the expected value of the Flory exponent of 0.6 for a polymer in a good solvent. The combined knowledge of how N(F)(-)(blob) varies with the fluorescence lifetime of the chromophore and the coil density of the polymer was used to propose a new means of studying coil-to-globule transitions with potential implications for predicting the rate of protein folding.     

Comparison of the association level of a pyrene-labeled associative polymer obtained from an analysis based on two different models

A hydrophobically modified alkali swellable emulsion copolymer (HASE) was labeled with pyrene and its fluorescence behavior was monitored by steady-state and time-resolved fluorescence as increasing amounts of the surfactant sodium dodecyl sulfate (SDS) were added to the solution. In aqueous solution, the pyrene pendants are aggregated. As SDS is added, the surfactant binds to the pyrene aggregates, which leads to their breakup at an onset SDS concentration of 1.25 x 10(-3) mol/L. The breakup of the pyrene aggregates is complete at 4.25 x 10(-3) mol/L, which is slightly larger than the critical micellar concentration of SDS in 0.01 M Na(2)CO(3) aqueous solution at pH 9 found to equal 3.5 x 10(-3) mol/L by surface tension measurements. The pyrene pendants were present as different species in solution, and the fractions representative of all emissive pyrene species were determined from the global analysis of the monomer and excimer fluorescence decays. Two analyses were applied to the decays. In the first analysis, the diffusional encounters between pyrene pendants were described by the blob model. In the second analysis, no assumptions were made on how the pyrene pendants encountered each other. Both analyses yielded identical results which demonstrate that the determination of the fractions of the different emissive pyrene species of a solution of a pyrene-labeled associative polymer does not depend on the model chosen to account for the diffusional encounters taking place between pyrene pendants.     

Chain conformations of poly(gamma-benzyl-L-glutamate) pre and post an electrospinning process

In order to reveal mechanisms for the electrospinning of proteins, this study focuses on the polymer chain conformation, which is considered to be a critical factor for successful electrospinning. Poly(gamma-benzyl-L-glutamate) (PBLG) is employed and the relationships between the chain conformations of the pre-spun PBLG molecules and the morphologies of the post-spun PBLG fibers are investigated. By combining viscosity measurements, and circular dichroism and FT-IR spectroscopies, chain conformations of the pre- and post-spun PBLG are characterized. The chain conformations of the pre-spun PBLG changes from an alpha-helix to a random coil upon changing the solvent ratios of dichloromethane (CH(2)Cl(2)) and trifluoroacetic acid (CF(3)COOH) from 100:0 to 0:100. In an alpha-helix conformation, the morphology of PBLG fibers is relatively thick, while that of the random coil is thin and homogenous. The mean fiber diameters decrease when the chain conformations change from an alpha-helix to a random coil. FT-IR spectroscopy and wide-angle X-ray diffraction measurements reveal that electrospinning predominantly induces an alpha-helical conformation in post-spun PBLG fibers, and more highly crystallized fibers are generated as the alpha-helical content in the pre-spun solution increases.     

Pyrene: a probe to study protein conformation and conformational changes

The review focuses on the unique spectral features of pyrene that can be utilized to investigate protein structure and conformation. Pyrene is a fluorescent probe that can be attached covalently to protein side chains, such as sulfhydryl groups. The spectral features of pyrene are exquisitely sensitive to the microenvironment of the probe: it exhibits an ensemble of monomer fluorescence emission peaks that report on the polarity of the probe microenvironment, and an additional band at longer wavelengths, the appearance of which reflects the presence of another pyrene molecule in spatial proximity (~10 ?). Its high extinction coefficient allows us to study labeled proteins in solution at physiologically relevant concentrations. The environmentally- and spatially-sensitive features of pyrene allow monitoring protein conformation, conformational changes, protein folding and unfolding, protein-protein, protein-lipid and protein-membrane interactions.     

Molar absorption coefficient of pyrene aggregates in water

The molar absorption coefficient of pyrene aggregates, epsilon(E0), was determined for a series of pyrene-labeled poly(N,N-dimethylacrylamide)s (Py-PDMA) having different pyrene contents. Aqueous solutions of Py-PDMA having pyrene contents ranging from 263 to 645 mumol of pyrene per gram of polymer were studied by UV-vis absorbance and time-resolved fluorescence spectroscopy. The global analysis of the monomer and excimer fluorescence decays with the fluorescence blob model yielded the fractions of the overall absorption contributed by all the pyrene species present in solution. The combined knowledge of the fractions obtained from the global analysis of the time-resolved fluorescence decays, the total absorption of the Py-PDMA solution obtained from UV-vis spectroscopy, and the total pyrene concentration in the solution obtained from the known pyrene content of each Py-PDMA sample led to the determination of the molar absorption coefficient of pyrene aggregates. Regardless of the pyrene content of the Py-PDMA samples and hence the level of association of the pyrene pendants in solution, all Py-PDMA samples yielded similar epsilon(E0) values over the range of wavelengths studied, namely, from 325 to 350 nm. The averaged epsilon(E0) was found to be red-shifted relative to unassociated pyrenes by 3 nm as well as having a value at the 0-0 peak of 21 000 M(-1).cm(-1) reduced from 34 700 M(-1).cm(-1) for unassociated pyrenes. The determination of epsilon(E0) enabled the first determination of the absolute fraction of associated pyrenes for aqueous solutions of a series of pyrene-labeled water-soluble polymers. The procedure outlined in this study is applicable to any pyrene-labeled water-soluble polymer and provides a new means to study quantitatively the effect of the hydrophilic-to-lipophilic balance on the hydrophobic associations generated by hydrophobically modified water-soluble polymers. As an application, the average number of pyrenes involved in a pyrene aggregate generated by Py-PDMA in water is determined.     

Monitoring folding transitions of synthetic,branched-chain polypeptides by capillary zone electrophoresis

The coil/helix transition of a synthetic, branched-chain polymeric polypeptide (poly (Lys(Glu(1)-DL-Ala(3))EAK), 50-Lys residues long in the backbone, as a function of increasing molarities of methanol in solution, is here studied by both, circular dichroism (CD) and capillary zone electrophoresis. CD spectra showed that, at 75% v/v methanol, the transition from random coil to fully helical structure was obtained, in a pH 1.1 HCI solution in the presence of 20 mM NaCI. CZE studies, run in parallel, exhibited the classical unfolding to folding sigmoidal transition, with mid-point at 60% v/v methanol concentration, plateauing at ca. 80% v/v organic solvent. Surprisingly, though, such unfolding to folding transition was accompanied by an expansion, rather than a contraction, of the resulting ordered polypeptide. As the charge of the polypeptide (a pure polycation at a pH of 2.1 in CZE) was kept rigorously constant, a plot of the radius of the polymer along the sigmoidal transition clearly showed that the radius of gyration of the helical, structured polypeptide was in fact larger than that of the random coil. Such results were confirmed by molecular dynamics simulations, which indicated that the dimensions of such polypeptide, in alpha-helix configuration, were 8.5 nm (in length) and 3.2 nm (in diameter), whereas those of the corresponding random coil were 7.2 nm (in length) and 5.1 nm (length of shorter axis). It would thus appear that the randomized structure assumes the shape of a more compact object, roughly resembling a "rugby ball".     

A case for using randomly labeled polymers to study long-range polymer chain dynamics by fluorescence

The process of excimer formation was studied for a series of pyrene end-labeled polystyrenes (PS(X)-Py 2 where X is the polymer molecular weight equal to 3, 4.5, 8, 12.7, and 14.6 K) and two series of polystyrenes randomly labeled with pyrene (CoE-PS and CoA-PS) in seven different solvents. The solvent viscosities ranged from 0.41 to 1.92 mPa x s, while the solvent quality ranged from good to poor solvents for polystyrene, as determined by intrinsic viscosity measurements. Steady-state fluorescence spectra of the pyrene-labeled polymers were acquired, and the excimer to monomer ratios showed that excimer formation increased strongly with a decrease in solvent viscosity. The monomer and excimer time-resolved fluorescence decays were also acquired and fitted globally to either the Birks' scheme or the fluorescence blob model (FBM) for the end- or randomly labeled polymers, respectively. All parameters reporting on the long-range polymer chain dynamics (LRPCD) obtained from the analysis of the fluorescence data acquired with the PS(X)-Py 2, CoE-PS, and CoA-PS series yielded virtually identical trends, demonstrating that these fluorescence experiments yield results that are internally consistent with one another. Considering the substantial advantages associated with the preparation and study of randomly labeled polymers, this report presents an appealing case for the use of randomly labeled polymers in the study of LRPCD.     

Photophysical and photochemical studies of chlorophyll a and cobalt(II)tetraphenylporphyrin in poly(L-glutamate)-decylammonium ion complex

Spectroscopic studies were carried out on chlorophyll a and cobalt(II)tetraphenylporphyrin solubilized in a poly(L-glutamate) (Poly(Glu))-decylammonium chloride (DeAC) complex system, in the presence of methylviologen (MV2+). The cooperative binding occurred between the anionic Poly(Glu) and the cationic DeAC, leading to the formation of micelle-like hydrophobic clusters of DeAC and also the change in conformation of the Poly(Glu) from the random coil to the alpha-helix. All of the absorption spectra, the fluorescence quantum yields and the fluorescence lifetimes indicated the existence of equilibrium between the aggregated biofunctional molecules in the bulk phase and the monomeric species in the complex phase of the Poly(Glu)-DeAC solution. The fluorescence quenching of the biofunctional molecules by methylviologen indicates that the conformation-dependent electron transfer occurs in the complex phase.     

Does adsorption at hydroxyapatite surfaces induce peptide folding? Insights from large-scale B3LYP calculations

Large-scale periodic quantum mechanical calculations (509 atoms, 7852 atomic orbitals) based on the hybrid B3LYP functional focused on the peptide folding induced by the adsorption on the (001) and (010) hydroxyapatite (HA) surfaces give interesting insights on the role of specific interactions between surface sites and the peptide, which stabilize the helix conformation over the "native" random coil ones for in silico designed model peptides. The two peptides were derived from the 12-Gly oligomer, with one (P1, C-tGGKGGGGGGEGGN-t) and two (P2, C-tGGKGGKEGGEGGN-t) glutamic acid (E) and lysine (K) residue mutations. The most stable gas-phase "native" conformation for both peptides resulted in a random coil (RC) structure, with the helix (H) conformation being ≈100 kJ mol(-1) higher in free energy. The two peptide conformations interact with the HA (001) and (010) surfaces by C═O groups via Ca(2+) ions, by hydrogen bond between NH(2) groups and the basic PO(4)(3-) groups and by a relevant fraction due to dispersion forces. Peptide adsorption was studied on the dry (001) surface, the wet one envisaging 2 H(2)O per surface Ca(2+) and, on the latter, also considering the adsorption of microsolvated peptides with 4 H(2)O molecules located at sites responsible of the interaction with the surface. The P1 mutant does prefer to be adsorbed as a random coil by ≈160 kJ/mol, whereas the reverse is computed for P2, preferring the helix conformation by ≈50 kJ/mol. Adsorption as helix of both P1 and P2 mutants brings about proton transfer toward the HA surfaces with a large charge transfer component to the interaction energy.     

UV resonance Raman determination of polyproline II, extended 2.5(1)-helix, and beta-sheet Psi angle energy landscape in poly-L-lysine and poly-L-glutamic acid

UV resonance Raman (UVR) spectroscopy was used to examine the solution conformation of poly-l-lysine (PLL) and poly-l-glutamic acid (PGA) in their non-alpha-helical states. UVR measurements indicate that PLL (at pH = 2) and PGA (at pH = 9) exist mainly in a mixture of polyproline II (PPII) and a novel left-handed 2.5(1)-helical conformation, which is an extended beta-strand-like conformation with Psi approximately +170 degrees and Phi approximately -130 degrees . Both of these conformations are highly exposed to water. The energies of these conformations are very similar. We see no evidence of any disordered "random coil" states. In addition, we find that a PLL and PGA mixture at neutral pH is approximately 60% beta-sheet and contains PPII and extended 2.5(1)-helix conformations. The beta-sheet conformation shows little evidence of amide backbone hydrogen bonding to water. We also developed a method to estimate the distribution of Psi Ramachandran angles for these conformations, which we used to estimate a Psi Ramachandran angle energy landscape. We believe that these are the first experimental studies to give direct information on protein and peptide energy landscapes.     

Microstructural reorganization and cargo release in pyrene urethane methacrylate random copolymer hollow capsules

We report the synthesis of polymer microcapsules by direct one-pot free radical random copolymerization approach. Urethane methacrylate comb monomers having pendant pyrene (Py) and 3-pentadecyl phenol (PDP) units were copolymerized in a random manner using benzoyl peroxide (BPO) as free radical initiator in dimethylformamide (DMF) as solvent. These copolymers and corresponding homopolymers spontaneously self-organized into microspheres upon drop casting from solvents like DMF and tetrahydrofuran (THF). Stable microspheres were obtained in water by dialyzing THF solution of the polymers against water in dialysis bags with molecular weight cutoff of ～2000. The hollow nature of the spheres was confirmed by rhodamine B (RhB) encapsulation followed by F?rster resonance energy transfer (FRET) based fluorescence emission from RhB upon exciting pyrene. The microenvironment inside the capsule was probed by following the I(1)/I(3) ratio of pyrene emission as well as RhB release as a function of temperature. The RhB encapsulated in the pyrene homopolymer PIHP-100Py capsules experienced strong donor-acceptor interaction and did not undergo complete release even at high temperature (85 °C). The encapsulated RhB from the copolymers with low pyrene incorporation was released almost fully upon heating beyond 50 °C. Pyrene moieties in the PIHP-100Py were shielded from surrounding water and experienced a hydrophobic environment, whereas in the low pyrene incorporated copolymer the PDP units were better shielded from the hydrophilic environment. This work represents a simple approach to produce polymer hollow capsules, and the varying pyrene incorporation was used to trace the microenvironment inside the capsules.     

Folding and translocation of the undecamer of poly-L-leucine across the water-hexane interface. A molecular dynamics study.

The undecamer of poly-L-leucine at the water-hexane interface is studied by molecular dynamics simulations. This represents a simple model relevant to folding and insertion of hydrophobic peptides into membranes. The peptide, initially placed in a random coil conformation on the aqueous side of the system, rapidly translocates toward the hexane phase and undergoes interfacial folding into an alpha-helix in the subsequent 36 ns. Folding is nonsequential and highly dynamic. The initially formed helical segment at the N-terminus of the undecamer becomes transiently broken and, subsequently, reforms before the remainder of the peptide folds from the C-terminus. The formation of intramolecular hydrogen bonds during the folding of the peptide is preceded by a dehydration of the participating polar groups, as they become immersed in hexane. Folding proceeds through a short-lived intermediate, a 3(10)-helix, which rapidly interconverts to an alpha-helix. Both helices contribute to the equilibrium ensemble of folded structures. The helical peptide is largely buried in hexane, yet remains adsorbed at the interface. Its preferred orientation is parallel to the interface, although the perpendicular arrangement with the N-terminus immersed in hexane is only slightly less favorable. In contrast, the reversed orientation is highly unfavorable, because it would require dehydration of C-terminus carbonyl groups that do not participate in intramolecular hydrogen bonding. For the same reason, the transfer of the undecamer from the interface to the bulk hexane is also unfavorable. The results suggest that hydrophobic peptides fold in the interfacial region and, simultaneously, translocate into the nonpolar side of the interface. It is further implied that peptide insertion into the membrane is accomplished by rotating from the parallel to the perpendicular orientation, most likely in such a way that the N-terminus penetrates the bilayer.     

Solvent-dependent conformational and fluorescence change of an N-phenylbenzohydroxamic acid derivative bearing two pyrene moieties

The conformation of N-phenylbenzohydroxamic acid has been reported to change depending on the solvent properties. Here, we newly synthesized N-phenylbenzohydroxamic acid derivative, which contains two pyrene moieties separated from the phenyl rings by ethylene linkers. It showed solvent-dependent conformational change, and its fluorescence was also solvent-dependent, that is, only monomer fluorescence of pyrene was observed in DMSO or DMF, whereas the excimer fluorescence was observed in CH₂Cl₂ or CHCl₃. Thus, the structural characteristics could be converted to fluorescence change as output, which would be a good candidate as a fluorescent sensor.     

Conformation of Poly-(L-lysine) Containing Some Azoaromatic Side Chains

The pH dependence of the conformation of poly-(L-lysine) with 4.6 mol% of its side chain amino groups attached to an azo dye, 4′ -dimethyl aminoazobenzene-4-carboxylic acid, has been studied. Circular dichroic spectra showed that in acidic as well as in neutral media the polymer exists in the random coil conformation, like that of poly-(L-lysine). In basic medium the polypeptide acquires a β-structure, unlike poly-(L-lysine) which exists in an α-helical conformation.     

荧光猝灭研究芳醚树枝形聚合物构象

本工作分别合成了外围修饰一个芘基团和核心修饰一个芘基团两个系列的芳醚树枝形聚合物Py-Gn-OH和Gn-CH₂-Py(n=1~4)。Py-Gn-OH和Gn-CH₂-Py的发光随代数增加而增强,荧光寿命增加。荧光猝灭实验结果表明,树枝形聚合物Py-Gn-OH和Gn-CH₂-Py的双分子猝灭速率常数均随代数增加而减小,表明树枝形聚合物发生了构象折叠,位于核心和外围的芘基团均被树枝形聚合物骨架包裹,随代数增加树枝形聚合物骨架增大,对芘基团包裹作用增强,导致猝灭剂接近芘基团的位阻增大。Gn-CH₂-Py体系的双分子猝灭速率常数均比相应代数Py-Gn-OH体系略小,说明树枝形聚合物骨架对连接在核心的芘基团的包裹程度比对连接在外围的芘基团略强。本工作为新型功能芳醚树枝形聚合物设计和应用提供了参考。     

The studies of FT-IR and CD spectroscopy on catechol oxidase I from tobacco

A novel copper-containing enzyme named COI (catechol oxidase I) has been isolated and purified from tobacco by extracting acetone-emerged powder with phosphate buffer, centrifugation at low temperature, ammonium sulfate fractional precipitation, and column chromatography on DEAE-sephadex (A-50), sephadex (G-75), and DEAE-celluse (DE-52). PAGE, SDS-PAGE were used to detect the enzyme purity, and to determine its molecular weight. Then the secondary structures of COI at different pH, different temperatures and different concentrations of guanidine hydrochloride (GdnHCl) were studied by the FT-IR, Fourier self-deconvolution spectra, and circular dichroism (CD). At pH 2.0, the contents of both alpha-helix and anti-parallel beta-sheet decrease, and that of random coil increases, while beta-turn is unchanged compared with the neutral condition (pH 7.0). At pH 11.0, the results indicate that the contents of alpha-helix, anti-parallel beta-sheet and beta-turn decrease, while random coil structure increases. According to the CD measurements, the relative average fractions of alpha-helix, anti-parallel beta-sheet, beta-turn/parallel beta-sheet, aromatic residues and disulfide bond, and random coil/gamma-turn are 41.7%, 16.7%, 23.5%, 11.3%, and 6.8% at pH 7.0, respectively, while 7.2%, 7.7%, 15.2%, 10.7%, 59.2% at pH 2.0, and 20.6%, 9.5%, 15.2%, 10.5%, 44.2% at pH 11.0. Both alpha-helix and random coil decrease with temperature increasing, and anti-parallel beta-sheet increases at the same time. After incubated in 6 mol/L guanidine hydrochloride for 30 min, the fraction of alpha-helix almost disappears (only 1.1% left), while random coil/gamma-turn increases to 81.8%, which coincides well with the results obtained through enzymatic activity experiment.     

Effect of urea on the conformational behavior of poly(N-isopropylacrylamide)

The effect of urea on the conformational behavior of poly(N-isopropylacrylamide) (PNIPAM) in dilute aqueous solution has been investigated using fluorescence spectroscopy, fluorescence quenching and fluorescence anisotropy measurements via pyrene (Py) probe and acenaphthylene (ACE) label studies. It was demonstrated that urea promotes the partitioning of the hydrophobic probe, Py, towards the bulk aqueous phase at temperatures above the lower critical solution temperature (LCST) of the polymer due to swelling of the compact coil conformation. However, the compact coil structure of the polymer at temperatures greater than its LCST is not completely destroyed, even for urea concentrations up to 3 M, at which the phase transition is hardly observed. As expected, urea has little effect on the conformational behavior of PNIPAM at temperatures below its LCST. Received: 9 February 2000/Accepted: 13 June 2000     

Statics and dynamics of dense polymer systems studied by monte carlo simulation

Monte Carlo simulations of coarse–grained models of macromolecules offer a unique tool to study the interplay between coil conformations, thermodynamic properties, and chain configurational relaxation and diffusion. Two examples are discussed where the chain conformation strongly differs from a gaussian coil: (i) collapsed chains in a bad solvent, where anomalous diffusion occurs in the Rouse limit and the relaxation time increases at least with the third power of chain length. (ii) Expulsion of a chain from a semidilute polymer brush. The initially stretched chain contracts to a gaussian coil and the center of mass moves outward with constant velocity until it reaches the region of the “last blob” where crossover to diffusive behavior occurs.