Cellulose hydrolysis – the role of monocomponent cellulases in crystalline cellulose degradation

Changes in the molecular structure of cellulose during hydrolysis with four recombinant -1,4-glycanases from the cellulolytic bacterium Cellulomonas fimi were assessed and compared in an attempt to elucidate the mechanism of crystalline cellulose degradation. It was apparent that the two endoglucanases, Cel6A and Cel5A, degraded sigmacell cellulose differently; Cel5A liberated more soluble sugars (cellobiose and cellotriose) and significantly altered the molecular weight distribution, while Cel6A had a limited effect on the polymer size and liberated primarily cellobiose and glucose. Additionally, both endoglucanases slightly increased the crystallinity of cellulose. In contrast, the cellobiohydrolases, Cel6B and Cel48A, had no effect on cellulose molecular weight and liberated only cellobiose and cellotriose. However, Cel48A was shown to be effective at reducing the crystallinity of the cellulosic substrate, while Cel6B increased the crystallinity index. Synergistic hydrolysis using combinations of the different enzymes showed that, although the cellulose was extensively hydrolysed, the molecular structure of the substrate was similar to the original material. This phenomenon suggests that the actions of individual monocomponent enzymes are offset by the concurrent modification by the complementing enzymes during synergistic hydrolysis.     

The active site of cellobiohydrolase Cel6A from Trichoderma reesei: the roles of aspartic acids D221 and D175

Trichoderma reesei cellobiohydrolase Cel6A is an inverting glycosidase. Structural studies have established that the tunnel-shaped active site of Cel6A contains two aspartic acids, D221 and D175, that are close to the glycosidic oxygen of the scissile bond and at hydrogen-bonding distance from each other. Here, site-directed mutagenesis, X-ray crystallography, and enzyme kinetic studies have been used to confirm the role of residue D221 as the catalytic acid. D175 is shown to affect protonation of D221 and to contribute to the electrostatic stabilization of the partial positive charge in the transition state. Structural and modeling studies suggest that the single-displacement mechanism of Cel6A may not directly involve a catalytic base. The value of (D2O)(V) of 1.16 +/- 0.14 for hydrolysis of cellotriose suggests that the large direct effect expected for proton transfer from the nucleophilic water through a water chain (Grotthus mechanism) is offset by an inverse effect arising from reversibly breaking the short, tight hydrogen bond between D221 and D175 before catalysis.     

Quantification of DNA BI/BII backbone states in solution. Implications for DNA overall structure and recognition

The backbone states of B-DNA influence its helical parameters, groove dimensions, and overall curvature. Therefore, detection and fine characterization of these conformational states are desirable. Using routine NMR experiments on a nonlabeled B-DNA oligomer and analyzing high-resolution X-ray structures, we investigated the relationship between interproton distances and backbone conformational states. The three H2'i-H6/8i+1, H2' 'i-H6/8i+1, and H6/8i-H6/8i+1 sequential distances were found cross-correlated and linearly coupled to epsilon-zeta values in X-ray structures and 31P chemical shifts (deltaP) in NMR that reflect the interconversion between the backbone BI (epsilon-zeta < 0 degrees ) and BII (epsilon-zeta > 0 degrees) states. These relationships provide a detailed check of the NMR data consistency and the possibility to extend the set of restraints for structural refinement through various extrapolations. Furthermore, they allow translation of deltaP in terms of BI/BII ratios. Also, comparison of many published deltaP in solution to crystal data shows that the impact of sequence on the BI/BII propensities is similar in both environments and is therefore an intrinsic and general property of B-DNA. This quantification of the populations of BI and BII is of general interest because these sequence-dependent backbone states act on DNA overall structure, a key feature for DNA-protein-specific recognition.     

Computational study on nonenzymatic peptide bond cleavage at asparagine and aspartic acid

Nonenzymatic peptide bond cleavage at asparagine (Asn) and glutamine (Gln) residues has been observed during peptide deamidation experiments; cleavage has also been reported at aspartic acid (Asp) and glutamic acid (Glu) residues. Although peptide backbone cleavage at Asn is known to be slower than deamidation, fragmentation products are often observed during peptide deamidation experiments. In this study, mechanisms leading to the cleavage of the carboxyl-side peptide bond of Asn and Asp residues were investigated using computational methods (B3LYP/6-31+G**). Single-point solvent calculations at the B3LYP/6-31++G** level were carried out in water, utilizing the integral equation formalism-polarizable continuum (IEF-PCM) model. Mechanism and energetics of peptide fragmentation at Asn were comparatively analyzed with previous calculations on deamidation of Asn. When deamidation proceeds through direct hydrolysis of the Asn side chain or through cyclic imide formationvia a tautomerization routeit exhibits lower activation barriers than peptide bond cleavage at Asn. The fundamental distinction between the mechanisms leading to deamidationvia a succinimideand backbone cleavage was found to be the difference in nucleophilic entities involved in the cyclization process (backbone versus side-chain amide nitrogen). If deamidation is prevented by protein three-dimensional structure, cleavage may become a competing pathway. Fragmentation of the peptide backbone at Asp was also computationally studied to understand the likelihood of Asn deamidation preceding backbone cleavage. The activation barrier for backbone cleavage at Asp residues is much lower (approximately 10 kcal/mol) than that at Asn. This suggests that peptide bond cleavage at Asn residues is more likely to take place after it has deamidated into Asp.     

Molecular weights of backbones in graft copolymers

Copolymerization of a monomer X with an existing polymer yields a copolymer contaminated by poly-X homopolymer and ungrafted polymer. Measurements on the pure copolymer can yield only the molecular weights of the cumulative backbone portion and of the cumulative graft portion. However, if the grafts can be isolated, then the molecular weights of their individual chains may be determined also. Since it is not isolatable, the backbone is not amenable to similar treatment. Equations are derived for the molecular weights (of any average) of individual backbone chains; they are expressed in terms of the following experimentally accessible quantities: (i) molecular weight of original polymer; (ii) molecular weight of ungrafted portion: and (iii) fraction by weight of the original polymer which remains ungrafted. The procedure is thus the analogue, applied to the backbone, of chemical isolation applied to the grafts. The treatment has been examined for copolymers prepared by grafting acrylic acid to nylon-6 by mutual irradiation.     

The Radical Cationic Repair Pathway of Cyclobutane Pyrimidine Dimer: The Effect of Sugar‐Phosphate Backbone

Radical cationic repair process of cis–syn thymine dimer has been investigated when (1) sugar‐phosphate backbones were substituted by hydrogen atoms, (2) phosphate group was substituted by two hydrogen atoms each on a sugar ring and (3) sugar‐phosphate backbone was taken into account. The effect of the interactions between N1 and N1′ lone pairs and the C6‐C6′ antibonding orbital are the most important evidences for the cleavage of the C6‐C6′ bond in the first step of radical cationic repair mechanism in the absence of the sugar‐phosphate backbone. The impact of the N1 and N1′ lone pairs on the C6‐C6′ bond cleavage decreases and the energy barrier of the cleavage of that bond significantly increases in the presence of the deoxynucleoside sugars and the sugar‐phosphate backbone.     

An approach to a chiral cycloalkanone-mediated asymmetric epoxidation of stilbene with oxone.

Chiral and C2-symmetric seven-membered cycloalkanones 2--6 bearing 1,2-diphenylethane-1,2-diamine and cyclohexane-1,2-diamine backbones were synthesized and evaluated their asymmetry inductive behaviours in an asymmetric epoxidation of stilbene with oxone. Although the reaction of the ketones 2 and 3 of a 1,2-diphenylethane-1,2-diamine backbone gave stilbene oxide in trace to 31% yield, those of the ketones 4-6 of a cyclohexane-1,2-diamine backbone gave the epoxide in satisfactorily high yield up to 98%. It is noteworthy that both reactions with use of stoichiometric and substoichiometric amounts of a ketone 4 gave the epoxide in the essentially same enantioselectivity, 17 and 18%. Eleven-membered cyclic ketones 7 and 8 bearing a binaphthalene backbone were also synthesized and examined their behaviours, while the enantioselectivity turned out to be marginal.     

Charge carrier mobilities in liquid crystalline mesomorphic poly(DI-n-Alkylsilylene)s; influence of backbone conformation

The charge transport properties of a series of symmetrically substituted mesomorphic poly(di-n-alkylsilylene)s are studied using the pulse-radiolysis time resolved microwave conductivity (PR-TRMC) technique. The observed conductivities for these polymers could be correlated with different backbone conformations present both in the crystalline solid phase and in the liquid crystalline mesophase. The transition from the solid phase to the mesophase is accompanied by a disordering of the silicon backbone that results in a decrease of the conductivity of up to two orders of magnitude. The charge carrier mobilities found varied from 5×10⁻⁵ m² /Vs for the all-trans conformation in the solid phase to 6×10⁻⁷ m² /Vs for the disordered backbone conformation in the mesophase. The anisotropic radiation-induced conductivity observed for aligned poly(di-n-hexylsilylene) samples demonstrate that charge carrier migration takes place preferentially in the direction of the polymer backbone.     

Characterization of maleimide dimers in photo-cross-linked copolyimide films

Copolyimide membranes are established materials for the separation of gaseous and liquid mixtures. Cross-linking of the polymer strands improves the physical and chemical stability. The photo-cross-linking of a 6FDA-ODA/6FDA-DABA 4?:?1 copolyimide membrane containing maleimide side groups as linker was investigated by FTIR spectroscopy. IR absorption spectra of the copolyimide backbone, 3-hydroxypropyldimethyl maleimide and the copolyimide functionalized with 3-hydroxypropyldimethyl maleimide were measured before and after different irradiation times and compared to each other. For band assignment a normal mode analysis was performed. The backbone of the polymer and the maleimide linker can be well distinguished due to their different spectral band positions. Only the films containing a maleimide moiety perform a photoreaction, the polymer backbone does not interfere. Based on the difference spectra and the results of the DFT calculations it was shown that the trans- and the cis-cycloadduct as well as the previously suggested 2-2'-adduct without a cyclobutane ring are formed upon UV irradiation. Evidence for an oxetane-like photoproduct was not found. Different time constants for the increase of the product bands were observed. The cycloadduct accumulates with a shorter time constant (τ = 2 to 5 min) than the 2-2'-adduct (τ = 75 min). The yield of the photo-cross-linking reaction was determined by spectral deconvolution and kinetic fitting of several marker bands. For the copolyimide synthesized in this work, a maximum value of 6% was reached. The stiffness of the copolyimide backbone inhibits further photo-cross-linking.     

Backbone conformational preferences and pseudorotational ring puckering of 1-aminocyclopentane-1-carboxylic acid

We have used quantum mechanical calculations at the B3LYP/6-311G(d,p) level to determine the conformational preferences of the N-acetyl-N'-methylamide derivative of 1-aminocyclopentane-1-carboxylic acid in the gas phase, chloroform solution, and water solution. The backbone conformation of this dipeptide has been described through the dihedral angles varphi and psi, while the pseudorotational phase angle was used to define the conformation of the cyclopentane ring. Results indicate that the backbone flexibility of this amino acid is restricted by the cyclic nature of the side chain, the relative stability of the different conformations depending on the polarity of the environment. The potential energy of the pseudorotation was also studied as a function of the backbone conformation. Interestingly, the conformation of the cyclic side chain depends on the backbone arrangement. Furthermore, the number of pseudorotational states accessible at room temperature is high in all the investigated environments, especially in aqueous solution. Finally, a set of force-field parameters for classical molecular mechanics calculations was developed for the investigated amino acid. Molecular dynamics simulations in both chloroform and aqueous solutions were performed to demonstrate the reliability of such parameters.     

On the consequences of side chain flexibility and backbone conformation on hydration and proton dissociation in perfluorosulfonic acid membranes

The flexibility of the side chain and effects of conformational changes in the backbone on hydration and proton transfer in the short-side-chain (SSC) perfluorosulfonic acid fuel cell membrane have been investigated through first principles based molecular modelling studies. Potential energy profiles determined at the B3LYP/6-31G(d,p) level in the two pendant side chain fragments: CF(3)CF(-O(CF(2))(2)SO(3)H)-(CF(2))(7)-CF(-O(CF(2))(2)SO(3)H)CF(3) indicate that the largest CF(2)-CF(2) rotational barrier along the backbone is nearly 28.9 kJ mol(-1) higher than the minimum energy staggered trans conformation. Furthermore, the calculations reveal that the stiffest portion of the side chain is near to its attachment site on the backbone, with CF-O and O-CF(2) barriers of 38.1 and 28.0 kJ mol(-1), respectively. The most flexible portion of the side chain is the carbon-sulfur bond, with a barrier of only 8.8 kJ mol(-1). Extensive searches for minimum energy structures (at the B3LYP/6-311G(d,p) level) of the same polymeric fragment with 4-7 explicit water molecules reveal that the perfluorocarbon backbone may adopt either an elongated geometry, with all carbons in a trans configuration, or a folded conformation as a result of the hydrogen bonding of the terminal sulfonic acids with the water. These electronic structure calculations show that the fragments displaying the latter 'kinked' backbone possessed stronger binding of the water to the sulfonic acid groups, and also undergo proton dissociation with fewer water molecules. The calculations point to the importance of the flexibility in both the backbone and side chains of PFSA membranes to effectively transport protons under low humidity conditions.     

The N6-methyl group of adenine further increases the BI stability of DNA compared to C5-methyl groups

Methylated DNA bases are natural modifications which play an important role in protein-DNA interactions. Recent experimental and theoretical results have shown an influence of the base modification on the conformational behavior of the DNA backbone. MD simulations of four different B-DNA dodecamers (d(GC)(6), d(AT)(6), d(G(5mCG)(5)C), and d(A(T6mA)(5)T)) have been performed with the aim to examine the influence of methyl groups on the B-DNA backbone behavior. An additional control simulation of d(AU)(6) has also been performed to examine the further influence of the C5-methyl group in thymine. Methyl groups in the major groove (as in C5-methylcytosine, thymine, or N6-methyladenine) decrease the BII substate population of RpY steps. Due to methylation a clearer distinction of the BI substate stability between YpR and RpY (CpG/GpC or TpA/ApT) steps arises. A positive correlation between the BII substate population and base stacking distances is seen only for poly(GC). A methyl group added into the major groove increases mean water residence times around the purine N7 atom, which may stabilize the BI substate by improving the hydration network between the DNA backbone and the major groove. The N6-methyl group also forms a water molecule bridge between the N6 and O4 atoms, and thus further stabilizes the BI substate.     

Sandwich layer structure of a novel polyimide with methylene spacing groups in biphenyl side chain

The aggregation structure of a novel polyimide (PI-6) with six methylene flexible spacing groups in biphenyl side chain was investigated using polarized optical microscope (POM), wide/small-angle X-ray diffraction (WAXD/SAXD) and molecular simulation approach. Depending on increasing temperature, the sandwich layer aggregation structure of PI-6 was developed from the thermo reversible gel. The backbones are lamellar stacking with 11.5 Å thickness. The side chains are randomly packed into the backbone lamellar intervals and the width of this layer for both backbone and side chain is about 23.1 Å which is consistent with the simulation data.     

On the structure of the inverse kinematics map of a fragment of protein backbone

Loop closure in proteins requires computing the values of the inverse kinematics (IK) map for a backbone fragment with 2n > or = 6 torsional degrees of freedom (dofs). It occurs in a variety of contexts, e.g., structure determination from electron-density maps, loop insertion in homology-based structure prediction, backbone tweaking for protein energy minimization, and the study of protein mobility in folded states. The first part of this paper analyzes the global structure of the IK map for a fragment of protein backbone with 6 torsional dofs for a slightly idealized kinematic model, called the canonical model. This model, which assumes that every two consecutive torsional bonds C(alpha)--C and N--C(alpha) are exactly parallel, makes it possible to separately compute the inverse orientation map and the inverse position map. The singularities of both maps and their images, the critical sets, respectively, decompose SO(3) x R(3) into open regions where the number of IK solutions is constant. This decomposition leads to a constructive proof of the existence of a region in R(3) x SO(3) where the IK of the 6-dof fragment attains its theoretical maximum of 16 solutions. The second part of this paper extends this analysis to study fragments with more than 6 torsional dofs. It describes an efficient recursive algorithm to sample IK solutions for such fragments, by identifying the feasible range of each successive torsional dof. A numerical homotopy algorithm is then used to deform the IK solutions for a canonical fragment into solutions for a noncanonical fragment. Computational results for fragments ranging from 8 to 30 dofs are presented.     

新型光学活性含氰基三联苯液晶聚噻吩合成与分子构象

合成了一种含有长柔性间隔基和氰基三联苯液晶基元的发光性聚噻吩衍生物{—[thiopheneyl—CH2—COO—(CH2)6—O—terphenyl—CN]n—, PT(6)TPhCN}. 利用傅里叶变换红外(FT-IR)光谱、核磁共振(1HNMR)、差示扫描量热(DSC)仪、偏光显微镜(POM)、紫外-可见(UV-Vis)吸收光谱和荧光(PL)光谱对单体和聚合物的结构及性质进行了表征. 单体都呈现出良好的液晶性能, 由于长间隔基的存在, 聚合物PT(6)TPhCN也呈现出良好SmAd相. 氰基三联苯的存在还赋予了聚合物良好的光致发光性能, 同时, 长间隔基也有效地降低了分子间的相互作用, 进一步增强了聚合物的发光性能. 另外, 研究发现, 在未引入任何手性元素的情况下, 聚合物主链在圆二色(CD)光谱中还呈现出明显的Cotton 效应, 这可能是由于大体积液晶基元的位阻效应和取向作用, 液晶基元环绕主链进行取向的同时诱导聚噻吩主链在长程范围内呈螺旋取向.     

Intramolecular hydrogen-bond-assisted planarization of asymmetrically functionalized alternating phenylene-pyridinylene copolymers

We report on the synthesis and characterization of a series of asymmetrically functionalized amphiphilic polymers with alternating pi-donor units (e.g., substituted benzene) and pi-acceptor units (e.g., pyridine) along the polymer backbone. The purpose of our present work involves incorporation of functional groups along the main chain to form intrachain hydrogen bonds, which promote planarization of the polymer backbone, and to fine-tune the optical properties. The structure-property relationship of polymers P1-P6 was investigated by means of analytical methods, such as FTIR spectroscopy, 1H and 13C NMR spectroscopy, UV/Vis spectroscopy, fluorescence spectroscopy, gel permeation chromatography, thermogravimetric analysis, cyclic voltammetry, and X-ray powder diffraction. All polymers were soluble in common organic solvents, and the optical and fluorescence spectra of the polymers showed significant changes according to the formation (P4, P5) or absence (P6) of intramolecular hydrogen bonding along the polymer backbone. Moreover, the 2,6- or 3,5-linkage of the pyridine rings in P5 and P6, respectively, reduced the conjugation along the polymer backbone and this is reflected in their optical properties. The optical properties of the polymers were influenced by the addition of acid (P1-P6), base (P4-P6), and metal ions (e.g., Cu2+, Fe3+, Ag+, Ni2+, Pd2+, Mn2+, Zn2+, Mg2+, and Pr3+). Such polymers could be used in various applications, including sensors and stimuli-responsive displays.     

Effects of the biological backbone on stacking interactions at DNA-protein interfaces: the interplay between the backbone···π and π···π components

The (gas-phase) MP2/6-31G*(0.25) π···π stacking interactions between the five natural bases and the aromatic amino acids calculated using (truncated) monomers composed of conjugated rings and/or (extended) monomers containing the biological backbone (either the protein backbone or deoxyribose sugar) were previously compared. Although preliminary energetic results indicated that the protein backbone strengthens, while the deoxyribose sugar either strengthens or weakens, the interaction calculated using truncated models, the reasons for these effects were unknown. The present work explains these observations by dissecting the interaction energy of the extended complexes into individual backbone···π and π···π components. Our calculations reveal that the total interaction energy of the extended complex can be predicted as a sum of the backbone···π and π···π components, which indicates that the biological backbone does not significantly affect the ring system through π-polarization. Instead, we find that the backbone can indirectly affect the magnitude of the π···π contribution by changing the relative ring orientations in extended dimers compared with truncated dimers. Furthermore, the strengths of the individual backbone···π contributions are determined to be significant (up to 18 kJ mol(-1)). Therefore, the origin of the energetic change upon model extension is found to result from a balance between an additional (attractive) backbone···π component and differences in the strength of the π···π interaction. In addition, to understand the effects of the biological backbone on the stacking interactions at DNA-protein interfaces in nature, we analyzed the stacking interactions found in select DNA-protein crystal structures, and verified that an additive approach can be used to examine the strength of these interactions in biological complexes. Interestingly, although the presence of attractive backbone···π contacts is qualitatively confirmed using the quantum theory of atoms in molecules (QTAIM), QTAIM electron density analysis is unable to quantitatively predict the additive relationship of these interactions. Most importantly, this work reveals that both the backbone···π and π···π components must be carefully considered to accurately determine the overall stability of DNA-protein assemblies.     

Liquid‐Crystalline Mesogens Based on Cyclo[6]aramides: Distinctive Phase Transitions in Response to Macrocyclic Host–Guest Interactions

Producing macrocyclic mesogens that are responsive to guest encapsulation presents a significant challenge. Cyclo[6]aramides, a type of macrocycle with a hydrogen‐bond‐constrained backbone, exhibit thermotropic lamellar, discotic nematic, hexagonal, and rectangular columnar mesophases over a considerably wide temperature range, including at room temperature. Additionally, cyclo[6]aramides show unusual mesophase transitions from lamellar to hexagonal columnar phase mediated by macrocyclic host–guest (H–G) interactions between the macrocycles and alkylammonium salts. The phase transition, triggered by an organic guest engaging in H–G interactions with a macrocyclic cavity, provides a novel strategy for manipulating the properties of liquid‐crystalline materials. The crystal structure of a homologous cyclo[6]aramide reveals a disk‐shaped, near‐planar molecular backbone that facilitates intermolecular π–π stacking and leads to columnar assembly.     

A Monte Carlo simulation study of branched polymers

Monte Carlo simulations are presented for the static properties of highly branched polymer molecules. The molecules consist of a semiflexible backbone of hard-sphere monomers with semiflexible side chains, also composed of hard-sphere monomers, attached to either every backbone bead or every other backbone bead. The conformational properties and structure factor of this model are investigated as a function of the stiffness of the backbone and side chains. The average conformations of the side chains are similar to self-avoiding random walks. The simulations show that there is a stiffening of the backbone as degree of crowding is increased, for example, if the branch spacing is decreased or side chain length is increased. The persistence length of the backbone is relatively insensitive to the stiffness of the side chains over the range investigated. The simulations reproduce most of the qualitative features of the structure factor observed in experiment, although the magnitude of the stiffening of the backbone is smaller than in experiment.     

Aspartic acid side chain effect—Experimental and theoretical insight

Gas-phase H/D exchange and density functional theory study of the Asp and Glu side-chain carboxylic group intrinsic reactivity is reported. H/D exchange site specific treatment and some additional theoretical calculations showed that a side-chain carboxylic group may initiate proton transfer along with bond formation to one of its oxygens, i.e., possibility to initiate selective of cleavage peptide bond ("aspartic acid effect"). That finding is used to select aspartic acid cleavage mechanisms (side-chain proton transfer either to backbone carbonyl or to amide nitrogen) for further computational study. B3LYP/6-31G(d) and G3(MP2)//B3LYP potential energy profiles of both mechanisms on a model system CH3CO-Asp-NHCH3 were constructed. Although energy employed in low-energy collision induced dissociation suffices for both mechanisms thresholds, energy transferred to specific modes suggests a complex one-step mechanism of proton transfer (from the side-chain carboxylic group to the backbone amide group), bond formation (between deprotonated carboxylic group and carbon atom of the backbone carbonyl), and peptide bond cleavage as favorable.