Gel formation and low-temperature intramolecular conformation transition of a triple-helical polysaccharide lentinan in water

The gelation behavior of the triple-helical polysaccharide lentinan fractions having different molecular weights in water at 25 degrees C were studied by using a rheometer. The analysis of concentration and molecular weight dependence of shear stress and shear viscosity showed that aqueous lentinan is a typical shear-thinning fluid, possessing potential as a viscosity control agent, and that a weak gel with entangled network structure formed. The dynamic oscillatory behavior of lentinan in the temperature range of 1-15 degrees C was also investigated by rheologic method. The storage modulus G' and complex viscosity eta* increased first with decreasing temperature, and underwent a maximum centered at 7-9 degrees C, and then decreased with further decreasing temperature. This abnormal phenomenon was ascribed to formation of rigid structure in the gel state, which was confirmed by the experimental results from micro-DSC. The micro-DSC curves showed that an endothermic peak appeared at 7-9 degrees C for lentinan in water upon heating, which was attributable to the intramolecular order-disorder structure transition similar to triple-helical polysaccharide schizophyllan. Namely, at lower temperature, the side glucose residues of lentinan (triplix II) formed a well-organized triple-helical structure (triplix I) through hydrogen-bonding with the surrounding water molecules. Moreover, this conformation transition was proved to be thermally reversible. (c) 2008 Wiley Periodicals, Inc. Biopolymers 89: 852-861, 2008.This article was originally published online as an accepted preprint. The "Published Online" date corresponds to the preprint version. You can request a copy of the preprint by emailing the Biopolymers editorial office at biopolymers@wiley.com.     

Molecular dynamics studies of side chain effect on the beta-1,3-D-glucan triple helix in aqueous solution

beta-1,3-D-glucans have been isolated from fungi as right-handed 6(1) triple helices. They are categorized by the side chains bound to the main triple helix through beta-(1-->6)-D-glycosyl linkage. Indeed, since a glucose-based side chain is water soluble, the presence and frequency of glucose-based side chains give rise to significant variation in the physical properties of the glucan family. Curdlan has no side chains and self-assembles to form an water-insoluble triple helical structure, while schizophyllan, which has a 1,6-D-glucose side chain on every third glucose unit along the main chain, is completely water soluble. A thermal fluctuation in the optical rotatory dispersion is observed for the side chain, indicating probable co-operative interaction between the side chains and water molecules. This paper documents molecular dynamics simulations in aqueous solution for three models of the beta-1,3-D-glucan series: curdlan (no side chain), schizophyllan (a beta-(1-->6)-D-glycosyl side-chain at every third position), and a hypothetical triple helix with a side chain at every sixth main-chain glucose unit. A decrease was observed in the helical pitch as the population of the side chain increased. Two types of hydrogen bonding via water molecules, the side chain/main chain and the side chain/side chain hydrogen bonding, play an important role in determination of the triple helix conformation. The formation of a one-dimensional cavity of diameter about 3.5 A was observed in the schizophyllan triple helix, while curdlan showed no such cavity. The side chain/side chain hydrogen bonding in schizophyllan and the hypothetical beta-1,3-D-glucan triple helix could cause the tilt of the main-chain glucose residues to the helix.     

Ordering in aqueous polysaccharide solutions. II. Optical rotation and heat capacity of aqueous solutions of a triple-helical polysaccharide schizophyllan

Deuterium oxide solutions of schizophyllan, a triple-helical polysaccharide, undergoing an order-disorder transition centered at 17 degrees C, were studied by optical rotation (OR) and heat capacity (C(p)) to elucidate the molecular mechanism of the transition and water structure in the solution and frozen states. The ordered structure at low temperature consisted of the side chains and water in the vicinity forming an ordered hydrogen-bonded network surrounding the helix core and was disordered at higher temperature. In the solution state appeared clearly defined transition curves in both the OR and C(p) data. The results for three samples of different molecular weights were analyzed theoretically, treating this transition as a typical linear cooperative transition from the ordered to disordered states and explained quantitatively if the molecular weight polydispersity of the sample was considered. The excess heat capacity C(EX)(p) defined as the C(p) minus the contributions from schizophyllan and D(2)O was estimated. In the frozen state it increased with raising temperature above 150 K until the mixture melted. This was compared with the dielectric increment observed in this temperature range and ascribed to unfreezable water. From the heat capacity and dielectric data, unfreezable water is mobile but more ordered than free water. In the solution state, the excess heat capacity originates from the interactions of D(2)O molecules as bound water and structured water, and so forth. Thus the schizophyllan triple helix molds water into various structures of differing orders in solution and in the solid state.     

Trifluoroethanol and binding to model membranes stabilize a predicted turn in a peptide corresponding to the first extracellular loop of the angiotensin II AT(1A) receptor

Deuterium oxide solutions of a triple-helical polysaccharide schizophyllan, undergoing an order-disorder transition centered around 17 degrees C, were studied by the time-domain reflectometry (TDR) to obtain dielectric dispersions in the solution and frozen states. In the solution state, the dispersion below the transition temperature is resolved in three dispersions (relaxation times at 0 degrees C) ascribed to side chain glucose residue (1; 102 ns), structured water (s; 2.0 ns) and bulk water (h), respectively, from low to high frequencies. Bulk water is divided into slow water (h2; 0.04 ns) and free or pure water (h1; 0.02 ns). Above the transition temperature structured water almost disappears and is compensated by slow water. Structured water is similar to bound water for proteins but different from it because of this transition behavior. Another dispersion (l) seen at the lowest frequency is assigned to the rotation of side-chain glucose residue coupled with hydrated water. Parts of this dispersion and structured water are suggested to constitute bound water. In the frozen state were observed a major dispersion (h; 0.14 ns) and a minor one (m; 28 ns), which were ascribed to considerably mobile and less mobile waters. They are similar to but not exactly the same as that for unfreezable water in bovine serum albumin solutions argued by Miura et al. (Biopolymers, 1995, Vol. 36, p. 9). Water is molded into different structures by the triple helix.     

Melting behaviour of schizophyllan extracellular polysaccharide gels in the temperature range between 5 and 20°C

Gels of the glucan schizophyllan, consisting of a 1,3-β- -linked backbone of glucose residues with 1,6-β- -glucosyl side groups, were found to show melting behaviour in the temperature range between 5 and 20°C, depending on the glucose concentration in the solvent (0–50 wt% glucose). While the qualitative features of the modulus-versus-concentration and modulus-versus-temperature rheological data for the gels can be modelled using modified cascade theory (which implicitly assumes that no sub-level of organisation exists in the gel structure), a consistent quantitative fit cannot be achieved. The inconsistencies found are consistent with the idea that the gel is composed of bundles (consisting of many triple helices of schizophyllan) with strong intra-bundle attraction and weak inter-bundle forces. Transmission Electron Microscopy (TEM) micrographs of diluted samples indicate that schizophyllan polymers engage in lateral aggregation of triple helical strands at temperatures below the melting temperature, suggesting that indeed bundles of polymers will be present in the gel state.     

The Contribution of Interchain Salt Bridges to Triple-Helical Stability in Collagen

Studies on collagen and collagen-like peptides suggest that triple-helical stability can vary along the amino acid chain. In this regard, it has been shown that lysine residues in the Y position and acidic residues in the X′ position of (GPO)₃GXYGX′Y′(GPO)₃ peptides lead to triple-helical structures with melting temperatures similar to (GPO)₈ (where O is hydroxyproline), which is generally regarded as the most stable collagen-like sequence of this length. This enhanced stability has been attributed to the formation of salt bridges between adjacent collagen chains. In this study, we explore the relationship between interchain salt bridge formation and triple-helical stability using detailed molecular simulations. Although our results confirm that salt bridges promote triple-helical stability, we find that not all salt bridges are created equal. In particular, lysine-glutamate salt bridges are most stabilizing when formed between residues in the middle strand (B) and the trailing strand (C), whereas lysine-aspartate salt bridges are most stabilizing when formed between residues in the leading (A) and middle (B) strand—the latter observation being consistent with recent NMR data on a heterotrimeric model peptide. Overall, we believe these data clarify the role of salt bridges in modulating triple-helical stability and can be used to guide the design of collagen-like peptides that have specific interchain interactions.     

Morphologies and conformation transition of lentinan in aqueous NaOH solution

Molecular morphologies and conformation transition of lentinan, a beta-(1-->3)-D-glucan from Lentinus edodes, were studied in aqueous NaOH solution by atomic force microscopy (AFM), viscometry, multiangle laser light scattering, and optical rotation measurements. The results revealed that lentinan exists as triple-helical chains and as single random-coil chains at NaOH concentration lower than 0.05M and higher than 0.08M, respectively. Moreover, the dramatic changes in weight-average molecular weight Mw, radius of gyration [s2](1/2), intrinsic viscosity [eta], as well as specific optical rotation at 589 nm [alpha]589 occurred in a narrow range of NaOH concentration between 0.05 and 0.08M NaOH, indicating that the helix-coil conformation transition of lentinan was carried out more easily than that of native schizophyllan and scleroglucan, and was irreversible. For the first time, we confirmed that the denatured lentinan molecule, which was dissolved in 0.15M NaOH to be disrupted into single coil chains, could be renatured as triple helical chain by dialyzing against abundant water in the regenerated cellulose tube at ambient temperature (15 degrees C). In view of the AFM image, lentinan in aqueous solution exhibited the linear, circular, and branched species of triple helix compared with native linear schizophyllan or scleroglucan.     

Towards a complete description of the structural and dynamic properties of the denatured state of barnase and the role of residual structure in folding

The detailed characterization of denatured proteins remains elusive due to their mobility and conformational heterogeneity. NMR studies are beginning to provide clues regarding residual structure in the denatured state but the resulting data are too sparse to be transformed into molecular models using conventional techniques. Molecular dynamics simulations can complement NMR by providing detailed structural information for components of the denatured ensemble. Here, we describe three independent 4 ns high-temperature molecular dynamics simulations of barnase in water. The simulated denatured state was conformationally heterogeneous with respect to the conformations populated both within a single simulation and between simulations. Nonetheless, there were some persistent interactions that occurred to varying degrees in all simulations and primarily involved the formation of fluid hydrophobic clusters with participating residues changing over time. The region of the beta(3-4) hairpin contained a particularly high degree of such side-chain interactions but it lacked beta-structure in two of the three denatured ensembles: beta(3-4) was the only portion of the beta-structure to contain significant residual structure in the denatured state. The two principal alpha-helices (alpha1 and alpha2) adopted dynamic helical structure. In addition, there were persistent contacts that pinched off core 2 from the body of the protein. The rest of the protein was unstructured, aside from transient and mostly local side-chain interactions. Overall, the simulated denatured state contains residual structure in the form of dynamic, fluctuating secondary structure in alpha1 and alpha2, as well as fluctuating tertiary contacts in the beta(3-4) region, and between alpha1 and beta(3-4), in agreement with previous NMR studies. Here, we also show that these regions containing residual structure display impaired mobility by both molecular dynamics and NMR relaxation experiments. The residual structure was important in decreasing the conformational states available to the chain and in repairing disrupted regions. For example, tertiary contacts between beta(3-4) and alpha1 assisted in the refolding of alpha1. This contact-assisted helix formation was confirmed in fragment simulations of beta(3-4) and alpha1 alone and complexed, and, as such, alpha1 and beta(3-4) appear to be folding initiation sites. The role of these sites in folding was investigated by working backwards and considering the simulation in reverse, noting that earlier time-points from the simulations provide models of the major intermediate and transition states in quantitative agreement with data from both unfolding and refolding experiments. Both beta(3-4) and alpha1 are dynamic in the denatured state but when they collide and make enough contacts, they provide a loose structural scaffold onto which further beta-strands pack. The beta-structure condenses about beta(3-4), while alpha1 aids in stabilizing beta(3-4) and maintaining its orientation. The resulting beta-structure is relatively planar and loose in the major intermediate. Further packing ensues, and as a result the beta-sheet twists, leading to the major transition state. The structure is still expanded and loops are not well formed at this point. Fine-tuning of the packing interactions and the final condensation of the structure then occurs to yield the native state.     

The folding mechanism of collagen-like model peptides explored through detailed molecular simulations

Collagen has a unique folding mechanism that begins with the formation of a triple-helical structure near its C terminus followed by propagation of this structure to the N terminus. To elucidate factors that affect the folding of collagen, we explored the folding pathway of collagen-like model peptides using detailed molecular simulations with explicit solvent. Using biased molecular dynamics we examined the latter stages of folding of a peptide model of native collagen, (Pro-Hyp-Gly)10, and a peptide that models a Gly --> Ser mutation found in several forms of osteogenesis imperfecta, (Pro-Hyp-Gly)3-Pro-Hyp-Ser-(Pro-Hyp-Gly)6. Starting from an unfolded state that contains a C-terminal nucleated trimer, (Pro-Hyp-Gly)10 folds to a structure where two of the three chains associate through water-mediated hydrogen bonds and the third is relatively separated from this dimer. Calculated free-energy profiles for folding from this intermediate to the final triple-helical structure suggest that further folding occurs at a rate of approximately one Pro-Hyp-Gly triplet per msec. In contrast, after 6 nsec of biased dynamics, the region N-terminal to the Ser residue in (Pro-Hyp-Gly)3-Pro-Hyp-Ser-(Pro-Hyp-Gly)6 folds to a structure where the three chains form close contacts near the N terminus, away from the mutation site. Further folding to an ideal triple-helical structure at the site of the mutation is unfavorable as the free energy of a triple-helical conformation at this position is more than 20 kcal/mol higher than that of a structure with unassociated chains. These data provide insights into the folding pathway of native collagen and the events underlying the formation of misfolded structures.     

Phosphorus-31 two-dimensional solid-state exchange NMR. Application to model membrane and biological systems.

Two-dimensional solid-state 31P NMR has been used to investigate the orientational exchange of phospholipids in gel and liquid-crystalline aqueous multilamellar dispersions and oriented multibilayers, and in biological membranes. In liquid-crystalline L alpha multilamellar dispersions, orientational exchange originates from the lateral diffusion of phospholipid molecules over the curved surface of the liposomes and is manifest by an increase in off-diagonal intensity, which correlates the 90 and 0 degrees orientations of the membrane normal with respect to the magnetic field when the system is fully exchanged. Spectral simulations of the time evolution of exchange allowed determination of the correlation times tau d for lateral diffusion. For DMPC and DPPC at comparable reduced temperatures, tau d values of 44 and 8 ms were obtained, respectively. The nature and rate of exchange observed for POPE at 30 degrees C is similar to that of DMPC at the same temperature. The measured correlation times are consistent with diffusion rates obtained by FRAP for liposomes with radii in the 1 micron range. In the gel phase of DPPC (30 degrees C), little orientational exchange is observed at mixing times up to 200 ms, demonstrating that the lateral diffusion is very slow. The correlation time for orientational exchange obtained from spectral simulations was approximately 900 ms; thus, exchange in the gel state is at least two orders of magnitude slower than in the liquid-crystalline state. In the P beta (ripple) phase, at temperatures between 34 and 39 degrees C, significant exchange is observed for mixing times between 50 and 200 ms. Exchange is also observed in oriented samples of DPPC in the P beta phase for mixing times of 50 ms, but not for oriented liquid-crystalline samples for mixing times up to 100 ms. The exchange observed in the ripple phase could originate from rapid lateral diffusion of "fast" diffusing phospholipid within defect structures, and/or from "slow" lateral diffusion of ordered phospholipid over the ripples. 2D experiments were also performed on pig erythrocyte ghosts and on intact pig spinal cord. Significant orientational exchange was observed with the erythrocyte ghosts at a mixing time of 200 ms, but almost no exchange was observed with the spinal cord at the same mixing time. Spectral simulations suggest tau d values of approximately 400 ms and 1.3 s for the erythrocyte ghosts and spinal cord at 30 degrees C.(ABSTRACT TRUNCATED AT 400 WORDS)     

Water structures of differing order and mobility in aqueous solutions of schizophyllan, a triple-helical polysaccharide as revealed by dielectric dispersion measurements

Dielectric dispersion measurements were made on aqueous solutions of a triple-helical polysaccharide schizophyllan over a wide concentration range 10-50 wt % at -45 to +30 degrees C. In the solution state, three different water structures with the different relaxation times tau were found, namely, bound water (taul), structured water (taus), and loosely structured water (tauls) in addition to free water (tauP). Structured water is less mobile and loosely structured water is nearly as mobile as free water, but bound water with taul is much less mobile, thus taul > taus > tauls greater, similar tauP. The order-disorder transition accompanies the conversion between structured water and loosely structured water. However, the species with taus remains even in the disordered state and constitutes part of bound water in the entire temperature range. In the frozen state, in addition to bulk water formed by partial melting, two mobile species existed, which were assigned to liquidlike bound water and found to be a continuation of bound water in the solution state. These relaxation time data are discussed in connection with the entropy levels of the four structures deduced from heat capacity data (cf. Yoshiba, K.; et al. Biomacromolecules 2003, 4, 1348-1356).     

Dissecting the assembly of Abeta16-22 amyloid peptides into antiparallel beta sheets

Multiple long molecular dynamics simulations are used to probe the oligomerization mechanism of Abeta(16-22) (KLVFFAE) peptides. The peptides, in the monomeric form, adopt either compact random-coil or extended beta strand-like structures. The assembly of the low-energy oligomers, in which the peptides form antiparallel beta sheets, occurs by multiple pathways with the formation of an obligatory alpha-helical intermediate. This observation and the experimental results on fibrillogenesis of Abeta(1-40) and Abeta(1-42) peptides suggest that the assembly mechanism (random coil --> alpha helix --> beta strand) is universal for this class of peptides. In Abeta(16-22) oligomers both interpeptide hydrophobic and electrostatic interactions are critical in the formation of the antiparallel beta sheet structure. Mutations of either hydrophobic or charged residues destabilize the oligomer, which implies that the 16-22 fragments of Arctic (E22G), Dutch (E22Q), and Italian (E22K) mutants are unlikely to form ordered fibrils.     

Interaction of the α2A domain of integrin with small collagen fragments

We here present a detailed study of the ligand-receptor interactions between single and triple-helical strands of collagen and the α2A domain of integrin (α2A), providing valuable new insights into the mechanisms and dynamics of collagen-integrin binding at a sub-molecular level. The occurrence of single and triple-helical strands of the collagen fragments was scrutinized with atom force microscopy (AFM) techniques. Strong interactions of the triple-stranded fragments comparable to those of collagen can only be detected for the 42mer triple-helical collagen-like peptide under study (which contains 42 amino acid residues per strand) by solid phase assays as well as by surface plasmon resonance (SPR) measurements. However, changes in NMR signals during titration and characteristic saturation transfer difference (STD) NMR signals are also detectable when α2A is added to a solution of the 21mer single-stranded collagen fragment. Molecular dynamics (MD) simulations employing different sets of force field parameters were applied to study the interaction between triple-helical or single-stranded collagen fragments with α2A. It is remarkable that even single-stranded collagen fragments can form various complexes with α2A showing significant differences in the complex stability with identical ligands. The results of MD simulations are in agreement with the signal alterations in our NMR experiments, which are indicative of the formation of weak complexes between single-stranded collagen and α2A in solution. These results provide useful information concerning possible interactions of α2A with small collagen fragments that are of relevance to the design of novel therapeutic A-domain inhibitors.     

Effects of pH and temperature on the structural and thermodynamic character of alpha-syn12 peptide in aqueous solution

The structural and thermodynamic characters of alpha-syn12 peptide in aqueous solution at different pH and temperatures have been investigated through temperature replica exchange molecular dynamics (T-REMD) simulations with GROMOS 43A1 force field. The two independent T-REMD simulations were completed at pH = 7.0 and 10.0, respectively. Each replica was run for 300 ns. The structural and thermodynamic characters of alpha syn12 peptide were studied based on the distributions of backbone dihedral angles, the free energy surface, and the stability of different type structure and the favorite conformations of the peptide. The results showed that the simulation at pH = 10.0 produced more sampling in alpha region than the simulation at pH = 7.0. The temperature changes from 283 K to 308 K result in negligible effects on the distributions of backbone dihedral angle. The beta hairpin conformation with Turn(9-6) and four hydrogen bonds (HB(4-11), HB(6-9), HB(9-6) and HB(11-4)) is the lowest free energy state in the simulation at pH = 7.0. However, for the simulation at pH = 10.0, the lowest free energy state corresponds to a structure with Turn(9-6) and two hydrogen bonds (HB(6-10) and HB(10-6)) induced by an overly strong residue-residue interaction effect between lysine residues. For the simulation at pH = 7.0, the free energy change of the alpha-syn12 peptide from the unfolded state to the beta hairpin state was in good agreement with the experiments and molecular dynamics simulation results for the other beta-peptides, the beta hairpin state of the alpha-syn12 peptide included the conformations that not only the Turn(9-6) is formed, but also the terminus are closed together in space. However, the subtle balances between lysine-lysine interactions and lysine-solvent interaction are disrupted in the simulation at pH = 10.0, which induced the assembly of lysine residues, the beta hairpin conformation is destabilized by the deprotonation of the Lys side chain. This study can help us to understand the conformation changes and the thermodynamic character of alpha;-syn12 peptide at atomic level induced by changing pH and temperature, which is propitious to reveal the nosogenesis of Parkinson disease. In our knowledge, this is the first report to study the influence of pH and temperature on isolated alpha-syn12 peptide in water by T-REMD.     

Static water structure detected by heat capacity measurements on aqueous solutions of a triple-helical polysaccharide schizophyllan

Heat capacity measurements were made on aqueous solutions of a triple-helical polysaccharide schizophyllan by precision adiabatic calorimetry over a wide range of concentrations 30.45-90.93 wt % at temperatures between 5 and 315 K. The heat capacity curves obtained were divided into four groups depending on the weight fraction of schizophyllan w regions I-IV. In region I, triple-helices with the sheath of bound water, structured water, and loosely structured water forming layers around the helix core are embedded in free water. In region II, there is no free water, and loosely structured water decreases until it vanishes, but structured water stays constant with increasing w. In region III, bound water remains unaffected, but structured water decreases with increasing w by overlapping each other. Finally, in region IV, only schizophyllan and bound water exist, the latter decreasing upon increasing w. The maximum thickness of each layer is 0.18(3) nm for bound water, 0.13(4) nm for structured water, and 0.23(6) nm for loosely structured water, and these layers of water are at the enthalpy levels of 53%, 93.7%, and nearly 100%, respectively, between ice (0%) and free water (100%).     

Initiation of spectrin dimerization involves complementary electrostatic interactions between paired triple-helical bundles

The spectrin heterodimer is formed by the antiparallel lateral association of an alpha and a beta subunit, each of which comprises largely a series of homologous triple-helical motifs. Initiation of dimer assembly involves strong binding between complementary motifs near the actin-binding end of the dimer. In this study, the mechanism of lateral spectrin association at this dimer nucleation site was investigated using the analytical ultracentrifuge to analyze heterodimers formed from recombinant peptides containing two or four homologous motifs from each subunit (alpha20-21/beta1-2; alpha18-21/beta1-4). Both the two-motif and four-motif dimer associations were weakened substantially with increasing salt concentration, indicating that electrostatic interactions are important for the dimer initiation process. Modeling of the electrostatic potential on the surface of the alpha20 and beta2 motifs showed that the side of the motifs comprising the A and B helices is the most favorable for association, with an area of positive electrostatic potential on the AB face of the beta2 motif opposite negative potential on the AB face of the alpha20 motif and vise versa. Protease protection analysis of the alpha20-21/beta1-2 dimer showed that multiple trypsin and proteinase K sites in the A helices of the beta2 and alpha21 motifs become buried upon dimer formation. Together, these data support a model where complementary long range electrostatic interactions on the AB faces of the triple-helical motifs in the dimer nucleation site initiate the correct pairing of motifs, i.e. alpha21-beta1 and alpha20-beta2. After initial docking of these complementary triple-helical motifs, this association is probably stabilized by subsequent formation of stronger hydrophobic interactions in a complex involving the A helices of both subunits and possibly most of the AB faces. The beta subunit A helix in particular appears to be buried in the dimer interface.     

Ultrasonic degradation of schizophyllan, an antitumor polysaccharide produced by Schizophyllum commune Fries

Schizophyllan, a water-soluble beta-D-glucan elaborated by Schizophyllum commune Fries, was partially depolymerized by ultrasonic irradiation to a low-molecular-weight polysaccharide, designated "sonic-degraded schizophyllan". Both native and degraded polysaccharides exhibited essentially the same antitumor activities against Sarcoma-180 ascites. Both glucans are comprised solely of D-glucose residues and have a main chain of (1 leads to 3)-beta-D-glucopyranosyl residues, one out of three glucose residues being attached as single, (1 leads to 6)-beta-D-glucopyranosyl groups. Although both glucans have similar structural features, significant differences are observed in such physical properties as molecular weight and intrinsic viscosity. End-group analysis by using radioisotope-labeled glucans suggests that ultrasonic degradation occurs mainly by cleavage of glycosidic bonds of the main chain of schizophyllan. The molecular weights of the native and sonic-degraded schizophyllan were shown to be 75% of those of corresponding, original schizophyllan preparations, suggesting that there is no anomalous linkage sensitive to periodate oxidation, and ultrasonic irradiation may cause random hydrolysis of (1 leads to 3)-beta-D-glucosidic linkages in the main chain.     

Successful molecular dynamics simulation of the zinc-bound farnesyltransferase using the cationic dummy atom approach

Farnesyltransferase (FT) inhibitors can suppress tumor cell proliferation without substantially interfering with normal cell growth, thus holding promise for cancer treatment. A structure-based approach to the design of improved FT inhibitors relies on knowledge of the conformational flexibility of the zinc-containing active site of FT. Although several X-ray structures of FT have been reported, detailed information regarding the active site conformational flexibility of the enzyme is still not available. Molecular dynamics (MD) simulations of FT can offer the requisite information, but have not been applied due to a lack of effective methods for simulating the four-ligand coordination of zinc in proteins. Here, we report in detail the problems that occurred in the conventional MD simulations of the zinc-bound FT and a solution to these problems by employing a simple method that uses cationic dummy atoms to impose orientational requirement for zinc ligands. A successful 1.0 ns (1.0 fs time step) MD simulation of zinc-bound FT suggests that nine conserved residues (Asn127alpha, Gln162alpha, Asn165alpha, Gln195alpha, His248beta, Lys294beta, Leu295beta, Lys353beta, and Ser357beta) in the active site of mammalian FT are relatively mobile. Some of these residues might be involved in the ligand-induced active site conformational rearrangement upon binding and deserve attention in screening and design of improved FT inhibitors for cancer chemotherapy.     

Comparison of the transition states for folding of two Ig-like proteins from different superfamilies

In the "fold approach" proteins with a similar fold but different sequences are compared in order to investigate the relationship between native state structure and folding behaviour. Here we compare the properties of the transition states for folding of TI I27, the 27th immunoglobulin domain from human cardiac titin, and that of TNfn3, the third fibronectin type III domain from human tenascin. Experimental phi-values were used as restraints in molecular dynamics simulations to determine the structures that make up the transition state ensembles (TSEs) for folding of the two proteins. The restrained simulations that we present allow a detailed structural comparison of the two TSEs to be made. Further calculations show explicitly that for both proteins the formation of the interactions involving the residues in the folding nucleus is sufficient for the establishment of the topology of the Ig-like fold. We found that, although the folding nuclei of the two proteins are similar, the packing of the folding nucleus of TI I27 is much tighter than that of TNfn3, reflecting the higher experimental phi-values and beta(T) (Tanford Beta) of TI I27. These results suggest that the folding nucleus can be significantly deformed to accommodate extensive sequence variation while conserving the same folding mechanism.     

Utilization of agricultural biomass in the production of the biopolymer schizophyllan

Schizophyllan is a homoglucan produced by the fungus Schizophyllum commune, with a β-1,3-linked backbone and β-1,6-linked side chains of single glucose units at every other residue. Schizophyllan is commercially produced for pharmaceutical and cosmetics uses. However, the unique physical properties of schizophyllan suggest that it may have biomaterials applications. Schizophyllan is conventionally produced by submerged culture fermentation using glucose as a carbon source. This study demonstrates for the first time the efficient utilization of agricultural biomass substrates, particularly distiller’s dried grains with solubles, for schizophyllan production. Sugar composition analysis, NMR, and permethylation linkage analysis confirmed that the recovered product was schizophyllan. Schizophyllan produced from agricultural residues was of a high molecular weight and exhibited solution viscosity properties similar to those of commercially produced material. Utilization of biomass substrates could reduce the cost of schizophyllan production and provide a new value-added bioproduct for integrated biorefineries of the future.