Graft copolymer composed of cationic backbone and bottle brush-like side chains as a physically adsorbed coating for protein separation by capillary electrophoresis

To stabilize electroosmotic flow (EOF) and suppress protein adsorption onto the silica capillary inner wall, a cationic hydroxyethylcellulose-graft-poly (poly(ethylene glycol) methyl ether methacrylate) (cat-HEC-g-PPEGMA) graft copolymer composed of cationic backbone and bottle brush-like side chains was synthesized for the first time and used as a novel physically adsorbed coating for protein separation by capillary electrophoresis. Reversed (anodal) and very stable EOF was obtained in cat-HEC-g-PPEGMA-coated capillary at pH 2.2-7.8. The effects of degree of cationization, PEGMA grafting ratio, PEGMA molecular mass, and buffer pH on the separation of basic proteins were investigated. A systematic comparative study of protein separation in bare and HEC-coated capillaries and in cat-HEC-g-PPEGMA-coated capillary was also performed. The basic proteins can be well separated in cat-HEC-g-PPEGMA-coated capillary over the pH range of 2.8-6.8 with good repeatability and high separation efficiency, because the coating combines good protein-resistant property of bottle brush-like PPEGMA side chains with excellent coating ability of cat-HEC backbone. Besides its success in separation of basic proteins, the cat-HEC-g-PPEGMA coating was also superior in the fast separation of other protein samples, such as protein mixture, egg white, and saliva, which indicates that it is a promising coating for further proteomics analysis.     

Role of aromatic side chains in the folding and thermodynamic stability of integral membrane proteins

Aromatic residues are frequently found in helical and beta-barrel integral membrane proteins enriched at the membrane-water interface. Although the importance of these residues in membrane protein folding has been rationalized by thermodynamic partition measurements using peptide model systems, their contribution to the stability of bona fide membrane proteins has never been demonstrated. Here, we have investigated the contribution of interfacial aromatic residues to the thermodynamic stability of the beta-barrel outer membrane protein OmpA from Escherichia coli in lipid bilayers by performing extensive mutagenesis and equilibrium folding experiments. Isolated interfacial tryptophanes contribute -2.0 kcal/mol, isolated interfacial tyrosines contribute -2.6 kcal/mol, and isolated interfacial phenylalanines contribute -1.0 kcal/mol to the stability of this protein. These values agree well with the prediction from the Wimley-White interfacial hydrophobicity scale, except for tyrosine residues, which contribute more than has been expected from the peptide models. Double mutant cycle analysis reveals that interactions between aromatic side chains become significant when their centroids are separated by less than 6 A but are nearly insignificant above 7 A. Aromatic-aromatic side chain interactions are on the order of -1.0 to -1.4 kcal/mol and do not appear to depend on the type of aromatic residue. These results suggest that the clustering of aromatic side chains at membrane interfaces provides an additional heretofore not yet recognized driving force for the folding and stability of integral membrane proteins.     

Molecular orientation of aromatic polycarbonates containing fluorene side chains by polarized infrared spectroscopy and birefringence analysis

The molecular orientation of an aromatic polycarbonate containing fluorene side chains was investigated by polarized infrared spectroscopy and birefringence analyses. The copolymers were synthesized from 2,2‐bis(4‐hydroxyphenyl)propane (BPA), 9,9‐bis(4‐hydroxy‐3‐methylpheny)fluorene (BMPF), and phosgene by interfacial polycondensation. The 1449‐cm^?1 band of the uniaxially oriented films, stretched at the glass‐transition temperature (T_g) plus 5 °C, was assigned to various combinations of CC stretching and CH in‐plane bending vibrations in the fluorene ring, and the transition moment angle was estimated to be 90°. The intrinsic birefringence of aromatic polycarbonate films with BMPF molar ratios ranging from 0.5 to 1 was obtained with the 1449‐cm^?1 band. The copolymer was estimated to show zero intrinsic birefringence at the BMPF molar ratio of 0.75, and the BMPF homopolymer showed negative intrinsic birefringence. A linear relationship between the volume fraction of BMPF units and the intrinsic birefringence indicated that the two monomer units of BPA and BMPF in each copolymer were not independent, and an intrinsic birefringence could be defined even in the copolymer. The sign of the photoelastic coefficient in the homopolymer with BMPF units was positive. The different signs of the photoelastic coefficient and the intrinsic birefringence suggest that the fluorene side‐chain orientation induced by stress in the glass state is quite different from the orientation of the uniaxially oriented films stretched at T_g + 5 °C. © 2003 Wiley Periodicals, Inc. J Polym Sci Part B: Polym Phys 41: 1554–1562, 2003     

Synthesis of branched polypropylene with isotactic backbone and atactic side chains by binary iron and zirconium single‐site catalysts

This article reports the use of a binary single‐site catalyst system for synthesizing comb‐branched polypropylene samples having isotactic polypropylene (iPP) backbones and atactic polypropylene (aPP) side chains from propylene feedstock. This catalyst system consisted of the bisiminepyridine iron catalyst {[2‐ArN?C(Me)]₂C₅H₃N}FeCl₂ [Ar = 2,6‐C₆H₃(Me)₂] ( 1 ) and the zirconocene catalyst rac‐Me₂Si(2‐MeBenz[e]Ind)₂ZrCl₂ ( 2 ). The former in situ generated 1‐propenyl‐ended aPP macromonomer, whereas the latter incorporated the macromonomer into the copolymer. The effects of reaction conditions, such as the catalyst addition procedure and the ratio of 1 / 2 on the branching frequency, were examined. Copolymer samples having a branching density up to 8.6 aPP side chains per 1000 iPP monomer units were obtained. The branched copolymers were characterized by ¹³C NMR and differential scanning calorimetry. © 2003 Wiley Periodicals, Inc. J Polym Sci Part A: Polym Chem 41: 1152–1159, 2003     

The short-lived signaling state of the photoactive yellow protein photoreceptor revealed by combined structural probes

The signaling state of the photoactive yellow protein (PYP) photoreceptor is transiently developed via isomerization of its blue-light-absorbing chromophore. The associated structural rearrangements have large amplitude but, due to its transient nature and chemical exchange reactions that complicate NMR detection, its accurate three-dimensional structure in solution has been elusive. Here we report on direct structural observation of the transient signaling state by combining double electron electron resonance spectroscopy (DEER), NMR, and time-resolved pump-probe X-ray solution scattering (TR-SAXS/WAXS). Measurement of distance distributions for doubly spin-labeled photoreceptor constructs using DEER spectroscopy suggests that the signaling state is well ordered and shows that interspin-label distances change reversibly up to 19 ? upon illumination. The SAXS/WAXS difference signal for the signaling state relative to the ground state indicates the transient formation of an ordered and rearranged conformation, which has an increased radius of gyration, an increased maximum dimension, and a reduced excluded volume. Dynamical annealing calculations using the DEER derived long-range distance restraints in combination with short-range distance information from (1)H-(15)N HSQC perturbation spectroscopy give strong indication for a rearrangement that places part of the N-terminal domain in contact with the exposed chromophore binding cleft while the terminal residues extend away from the core. Time-resolved global structural information from pump-probe TR-SAXS/WAXS data supports this conformation and allows subsequent structural refinement that includes the combined energy terms from DEER, NMR, and SAXS/WAXS together. The resulting ensemble simultaneously satisfies all restraints, and the inclusion of TR-SAXS/WAXS effectively reduces the uncertainty arising from the possible spin-label orientations. The observations are essentially compatible with reduced folding of the I(2)' state (also referred to as the 'pB' state) that is widely reported, but indicates it to be relatively ordered and rearranged. Furthermore, there is direct evidence for the repositioning of the N-terminal region in the I(2)' state, which is structurally modeled by dynamical annealing and refinement calculations.     

Simultaneous construction of polymer backbone and side chains by three‐component polycondensation. Synthesis of polyethers with propargyl side chains from dialdehydes,alkylene bis(trimethylsilyl) ethers,and allenyltrimethylsilane

Polyethers with propargyl side chains were synthesized by the acid‐catalyzed reaction of dialdehydes 1 , alkylene bis(trimethylsilyl) ethers 2 , and allenyltrimethylsilane 4 . When ethylene glycol bis(trimethylsilyl) ether was used as 2 , only oligomer was obtained. However, the use of 2 with longer carbon chains gave the desired polyethers consisting of 1 , 2 , and 4 in the molar ratio 1:1:2 in good yields in the presence of 10 mol % triphenylmethyl perchlorate (TrClO₄) at ?20 °C. This polyether was treated with organometallic reagents such as Co complex or CuCl to give a cross‐linked polymer. This polymer synthesis is unusual in that it concurrently constructs both the polymer backbone and the functional side chains from three starting compounds. © 2005 Wiley Periodicals, Inc. J Polym Sci Part A: Polym Chem 43: 5440–5448, 2005     

Synthesis and characterization of graft copolymers based on poly(p-phenylene terephthalamide) backbone and well-defined polystyrene side chains

<正>A novel dualfunctional monomer,2-(2',2',6',6'-tetramethyl-piperidinyl-1'-oxy)methylbenzene-1,4-dioyl chloride hydrochloride,with two acid chloride groups for step-growth polymerization and a nitroxide group for the mediation of living radical polymerization was synthesized.It was first copolymerized with terephthaloyl chloride and p-phenylenediamine at a feed molar ratio of 1:3:4 in N-methyl-2-pyrrolidone containing 10 wt%calcium chloride at -10℃to yield a poly(p-phenylene terephthalamide) based macroinitiator,which initiated radical polymerization of styrene at 125℃to obtain a series of poly(p-phenylene terephthalamide)-g-polystyrenes.A combinatory analysis of proton nuclear magnetic resonance spectroscopy,Fourier transform infrared spectroscopy,elementary analysis,thermogravimetry and gel permeation chromatography indicated that the macroinitiator induced the radical polymerization of styrene to proceed in a well-controlled way.The molecular weight of side-chains increased with an increase of monomer conversion,and the molecular weight distribution index remained lower than 1.5.The graft copolymers showed a remarkably improved solubility in N-methyl -2-pyrrolidone and much depressed crystallinity in bulk.     

Dehydration in the folding of reduced cytochrome c revealed by the electron-transfer-triggered folding under high pressure

We determined the activation volume associated with protein folding of reduced cytochrome c from the collapsed intermediate to the native state. The folding rate was followed by a change in the absorption (420 nm) at various pressures between 0.1 and 200 MPa and at various concentrations of denaturant (guanidine hydrochloride) between 3.2 and 4.0 M. Dependence of the folding rate on both these factors revealed that the activation volume at ambient pressure in the absence of denaturant is negative (DeltaVf0 = -14 (+/-8) cm3.mol-1). Such a negative activation volume can be accounted for by a decrease in volume resulting from the dehydration of hydrophobic groups, primarily the heme group. Dehydration, which increases the entropy of the protein system, compensates for a decrease in the entropy accompanying the formation of the more compact and ordered transition state. We, therefore, propose that the positive change in the activation entropy for the folding reaction is due to the dehydration of hydrophobic groups. Furthermore, dehydration entropically promotes the protein folding reaction.     

LEAP: Highly accurate prediction of protein loop conformations by integrating coarse‐grained sampling and optimized energy scores with all‐atom refinement of backbone and side chains

Prediction of protein loop conformations without any prior knowledge (ab initio prediction) is an unsolved problem. Its solution will significantly impact protein homology and template‐based modeling as well as ab initio protein‐structure prediction. Here, we developed a coarse‐grained, optimized scoring function for initial sampling and ranking of loop decoys. The resulting decoys are then further optimized in backbone and side‐chain conformations and ranked by all‐atom energy scoring functions. The final integrated technique called loop prediction by energy‐assisted protocol achieved a median value of 2.1 Å root mean square deviation (RMSD) for 325 12‐residue test loops and 2.0 Å RMSD for 45 12‐residue loops from critical assessment of structure‐prediction techniques (CASP) 10 target proteins with native core structures (backbone and side chains). If all side‐chain conformations in protein cores were predicted in the absence of the target loop, loop‐prediction accuracy only reduces slightly (0.2 Å difference in RMSD for 12‐residue loops in the CASP target proteins). The accuracy obtained is about 1 Å RMSD or more improvement over other methods we tested. The executable file for a Linux system is freely available for academic users at http://sparks‐lab.org . © 2013 Wiley Periodicals, Inc.     

Behavior of thermosensitive graft copolymer with aromatic polyester backbone and poly-2-ethyl-2-oxazoline side chains in aqueous solutions

Thermoresponsive graft copolymers with alkylene-aromatic polyester main chain and poly-2-ethyl-2-oxazoline side chains were synthesized. Two copolymer samples which differed in grafting density (0.5 and 0.7) were studied using dynamic and static light scattering and turbidimetry in aqueous solutions at concentration 0.0053?g?cm^?3. Hydrodynamic radii of scattering objects and their contribution to light scattering were obtained as a function of temperature in a wide temperature interval. Temperatures of phase separation were found out. Effect of grafting density on the copolymer behavior in aqueous solutions upon heating was determined. In particular, the phase separation temperature reduces with the decreasing grafting density.     

Substrate binding and sequence preference of the proteasome revealed by active-site-directed affinity probes

Background: The proteasome is a multicatalytic protease complex responsible for most cytosolic protein breakdown. The complex has several distinct proteolytic activities that are defined by the preference of each for the carboxyterminal (P1) amino acid residue. Although mutational studies in yeast have begun to define substrate specificities of individual catalytically active β subunits, little is known about the principles that govern substrate hydrolysis by the proteasome.Results: A series of tripeptide and tetrapeptide vinyl sulfones were used to study substrate binding and specificity of the proteasome. Removal of the aromatic amino-terminal cap of the potent tripeptide vinyl sulfone proteasome inhibitor 4-hydroxy-3-iodo-2-nitrophenyl-leucinyl-leucinyl-leucine vinyl sulfone resulted in the complete loss of binding and inhibition. Addition of a fourth amino acid (P4) to the tri-leucine core sequence fully restored inhibitory potency. 1251-labeled peptide vinyl sulfones were also used to examine inhibitor binding and to determine the correlation of subunit modification with inhibition of peptidase activity. Changing the amino acid in the P4 position resulted in dramatically different profiles of β-subunit modification.Conclusions: The P4 position, distal to the site of hydrolysis, is important in defining substrate processing by the proteasome. We observed direct correlations between subunit modification and inhibition of distinct proteolytic activities, allowing the assignment of activities to individual β subunits. The ability of tetrapeptides, but not tripeptide vinyl sulfones, to act as substrates for the proteasome suggests there could be a minimal length requirement for hydrolysis by the proteasome. These studies indicate that it is possible to generate inhibitors that are largely specific for individual β subunits of the proteasome by modulation of the P4 and carboxy-terminal vinyl sulfone moieties.     

Understanding the role of the topology in protein folding by computational inverse folding experiments

Recent studies suggest that protein folding should be revisited as the emergent property of a complex system and that the nature allows only a very limited number of folds that seem to be strongly influenced by geometrical properties. In this work we explore the principles underlying this new view and show how helical protein conformations can be obtained starting from simple geometric considerations. We generated a large data set of C-alpha traces made of 65 points, by computationally solving a backbone model that takes into account only topological features of the all-alpha proteins; then, we built corresponding tertiary structures, by using the sequences associated to the crystallographic structures of four small globular all-alpha proteins from PDB, and analysed them in terms of structural and energetic properties. In this way we obtained four poorly populated sets of structures that are reasonably similar to the conformational states typical of the experimental PDB structures. These results show that our computational approach can capture the native topology of all-alpha proteins; furthermore, it generates backbone folds without the influence of the side chains and uses the protein sequence to select a specific fold among the generated folds. This agrees with the recent view that the backbone plays an important role in the protein folding process and that the amino acid sequence chooses its own fold within a limited total number of folds.     

Array of aromatic amino acid side chains located near the chromophore of photoactive yellow protein

The role of the array of aromatic amino acid side chains located close to the chromophore binding loop of photoactive yellow protein (PYP) was studied using the alanine-substitution mutagenesis. Phe92, Tyr94, Phe96 and Tyr98 were replaced with alanine (F92A, Y94A, F96A and Y98A, respectively), then these mutants were characterized by UV-visible absorption spectra, circular dichroism (CD) spectra, thermal stability and photocycle kinetics. Absorption maxima of F92A, Y94A, F96A and Y98A were 444, 442, 439 and 447 nm, respectively, different to wild type (WT) at 446 nm. Far-UV CD spectra of mutants other than F92A were different from WT, indicating that Tyr94, Phe96 and Tyr98 maintain the native secondary structure of PYP. Mid-point temperatures of thermal denaturation of F92A, Y94A and F96A, estimated by the CD signal at 222 nm, were 5-10 degrees C lower than WT. Time constants of the photocycle estimated by flash-induced absorbance change were 0.36 s for WT and 1.4 s for Y98A, however, 100, 30 and 3000 times slower than WT for F92A, Y94A and F96A, respectively. Tyr98 is located in the loop region, whereas Phe92, Tyr94 and Phe96 are incorporated in the beta4 strand, showing that aromatic amino acid residues in the beta-sheet regulate the absorption spectrum, thermal stability and photocycle of PYP. Aromatic rings of Phe92, Tyr94 and Phe96 lie nearly perpendicular to the aromatic ring of Phe75 or chromophore. Possible weak hydrogen bonds between the aromatic ring hydrogen and pi-electrons of these residues are discussed.     

Fine-tuning of polymer photovoltaic properties by the length of alkyl side chains

This paper reports the synthesis and characteristics of a series of alkyl-substituted planar polymers. The physical properties are carefully tuned to optimize their photovoltaic performance. Depending on the length of soluble alkyl side chains which modify the structural order and orientation substantially in polymer backbones, the device performance can be improved significantly. The tuning of HOMO energy levels optimized polymers’ spectral coverage of absorption and their hole mobility, as well as miscibility with fullerene; all these efforts enhanced polymer solar cell performances. The shortcircuit current, Jsc for polymer solar cells was increased by adjusting polymer chain packing ability. It was found that films with well distributed polymer/fullerene interpenetrating network exhibit improved solar cell conversion efficiency. Enhanced efficiency up to 5.8% has been demonstrated. The results provide important insights about the roles of flexile chains in structure-property relationship for the design of new polymers to be used in high efficient solar cells.     

Simultaneous construction of the polymer backbone and side chains by three‐component polycondensation: The synthesis of polyurethanes with allyl side chains from dialdehydes,alkylene N,N′‐bis(trimethylsilyl) carbamates,and allyltrimethylsilane

Polyurethanes with allyl side chains were synthesized by the simultaneous acid‐catalyzed reaction of dialdehydes ( 1 ), alkylene N,N′‐bis(trimethylsilyl) carbamates ( 4 ), and allyltrimethylsilane ( 5 ). When 5 was added to a mixture of 1 , 4 , and the catalyst, a low molecular weight polymer was formed, as well as a large amount of an insoluble gel. However, when a mixture of 1 , 4 , and 5 was added to the catalyst, the formation of gel was depressed, and the desired polyurethanes, consisting of 1 , 4 , and 5 in a molar ratio of 1/1/2, were obtained in good yields. This polyurethane synthesis is unusual in that it concurrently constructs both the polymer backbone and the functional side chains from three starting compounds. © 2002 Wiley Periodicals, Inc. J Polym Sci Part A: Polym Chem 40: 1236–1242, 2002