We recently demonstrated that the expression of the importin α subtype is switched from α2 to α1 during neural differentiation in mouse embryonic stem cells (ESCs) and that this switching has a major impact on cell differentiation. In this study, we report a cell-fate determination mechanism in which importin α2 negatively regulates the nuclear import of certain transcription factors to maintain ESC properties. The nuclear import of Oct6 and Brn2 was inhibited via the formation of a transport-incompetent complex of the cargo bound to a nuclear localization signal binding site in importin α2. Unless this dominant-negative effect was downregulated upon ESC differentiation, inappropriate cell death was induced. We propose that although certain transcription factors are necessary for differentiation in ESCs, these factors are retained in the cytoplasm by importin α2, thereby preventing transcription factor activity in the nucleus until the cells undergo differentiation.
Small-angle X-ray scattering profiles for an amylose fragment (maltoheptaose) in aqueous solution were observed and compared with the theoretical profiles calculated for an ensemble of chain conformations generated by molecular dynamics simulations and Monte Carlo simulations. The Monte Carlo results based on the disaccharide conformation energy map obtained without explicitly considering surrounding water molecules were in satisfactory agreement with the experimental results, provided that the effective dielectric constant was set to four. In contrast, the results of the fully solvated molecular dynamics simulations performed using the Cff91, Cff, Gromos, Glycam93, and Glycam99 force-fields were unexpectedly different from each other. Among them, Cff91 gave most satisfactory agreement with experiment, but the other fields yielded conformations that were somewhat or highly extended. It was also shown that recently developed Glycam99 is a significant improvement over Glycam 93. The representative snapshots of the two successful simulations resembled the regular helical structure reported by Goldsmith et al. (J. Mol. Biol. 1982, 156, 411). The source of the large force-field dependence was investigated by examining the various Ramachandran-like plots for the glycosidic torsion angles. For comparison, similar plots of ab initio energy for maltose (i.e., a fragment with two glucose units) were also calculated at the Hartree-Fock level, although in a simplified manner. These plots suggest that the extended conformation arises from too strong a preference for a certain rotational isomeric state of the glycosidic linkage. A procedure to remedy this over-preference can be devised, although a need of further elaboration of the force-field is indicated. The significance of force-fields is also illustrated in modeling a cyclodextrin composed of 14 glucose units.
The fast multipole method proposed by Greengard and Rokhlin (GR) is applied to large biomacromolecular systems. In this method, the system is divided into a hierarchy of cells, and electric field exerted on a particle is decomposed into two parts. The first part is a rapidly varying field due to nearby cells, so that it needs rigorous pairwise calculations. The second part is a slowly varying local field due to distant cells; hence, it allows rapid calculations through a multipole expansion technique. In this work, two additional possibilities for improving the performance are numerically examined. The first is an improvement of the convergence of the expansion by increasing the number of nearby cells, without including higher-order multipole moments. The second is an acceleration of the calculations by the particle-particle and particle-mesh/multipole expansion (PPPMiMPE) method, which uses fast Fourier transform instead of the hierarchy. For this purpose, the PPPM/MPE method originally developed by the authors for a periodic system is extended to a nonperiodic isolated system. The advantages and disadvantages of the GR and PPPMiMPE methods are discussed for both periodic and isolated systems. It is numerically shown that these methods with reasonable costs can reduce the error in potential felt by each particle to 0.1-1 kcalimol, much smaller than the 30-kcal/mol error involved in conventional simple truncations. 0 1994 by John Wiley & Sons, Inc. ment of long-range electrostatic interactions. Various efficient methods'-" have been proposed to overcome the insufficiencv of conventional simule
The substrate specificity and the transglycosylation activity of neopullulanase was altered by site-directed mutagenesis on the basis of information from a threedimensional structure predicted by computer-aided molecular modeling. According to the predicted three-dimensional structure of the enzyme-substrate complex, it was most likely that Ile-358 affected the substrate preference of the enzyme. Replacing Ile-358 with Trp, which has a bulky side chain, reduced the acceptability of ␣-(136)-branched oligo-and polysaccharides as substrates. The characteristics of the I358W-mutated enzyme were quite different from those of wild-type neopullulanase and rather similar to those of typical starch-saccharifying ␣-amylase. In contrast, replacing Ile-358 with Val, which has a smaller side chain, increased the preference for ␣-(136)-branched oligosaccharides and pullulan as substrates. The transglycosylation activity of neopullulanase appeared to be controlled by manipulating the hydrophobicity around the attacking water molecule, which is most likely used to cleave the glucosidic linkage in the hydrolysis reaction. We predicted three residues, Tyr-377, Met-375, and Ser-422, which were located on the entrance path of the water molecule might be involved. The transglycosylation activity of neopullulanase was increased by replacing one of the three residues with more hydrophobic amino acid residues; Y377F, M375L, and S422V. In contrast, the transglycosylation activity of the enzyme was decreased by replacing Tyr-377 with hydrophilic amino acid residues, Asp or Ser.
It has recently been discovered that cycloamylose, cyclic α-1,4-glucan with a degree of polymerization ranging from 17 to several hundred, can be produced by the action of potato d-enzyme (EC 2.4.1.25) on amylose. To obtain structural insights into this new series of cycloamylose, the topological aspects of its circular structure were discussed in terms of a ribbon model properly introduced. A simple analysis of the chain-length dependence of the product population was performed on the basis of an elastic wire model. If the amylose chain is rather stiff, a double-helical structure with foldbacks is more reasonable than a circularized single-helical structure. This is because theoretical calculations for the latter predict a periodic population oscillation, which is caused by the need for the helix ends to meet, whereas such an oscillation was not experimentally observed. However, due to the unknown chain stiffness, it is difficult to draw a definite conclusion. To answer some questions that arose in the above analysis, atomistic models are necessary. Therefore, crude atomistic models were created in close analogy with the modeling of supercoiled DNA. The models were then refined by molecular dynamics simulation performed with a fast multipole method. Both structures formed a hollow tube with internal diameter of 5−6 Å. As far as the enthalpic contribution is concerned, the simulation results support the double-helical structure. The simulation results also indicated appreciable chain flexibility, which might play an important role in accommodating guest molecules.
To make improved treatments of electrostatic interactions in biomacromolecular simulations, two possibilities are considered. The f i s t is the famous particle-particle and particle-mesh (PPPM) method developed by Hockney and Eastwood, and the second is a new one developed here in their spirit but by the use of the multipole expansion technique suggested by Ladd. It is then numerically found that the new PPPM method gives more accurate results for a two-particle system at small separation of particles. Preliminary numerical examination of the various computational methods for a single configuration of a model BPTI-water system containing about 24,000 particles indicates that both of the PPPM methods give far more accurate values with reasonable computational cost than do the conventional truncation methods. It is concluded the two PPPM methods are nearly comparable in overall performance for the many-particle systems, although the fist method has the drawback that the accuracy in the total electrostatic energy is not high for configurations of charged particles randomly generated. 0 1993 by John Wiley & Sons. Inc.
Amylomaltase from Thermus aquaticus catalyzes intramolecular transglycosylation of ␣-1,4 glucans to produce cyclic ␣-1,4 glucans (cycloamyloses) with degrees of polymerization of 22 and higher. Although the amylomaltase mainly catalyzes the transglycosylation reaction, it also has weak hydrolytic activity, which results in a reduction in the yield of the cycloamyloses. In order to obtain amylomaltase with less hydrolytic activity, random mutagenesis was perfromed for the enzyme gene. Tyr54 (Y54) was identified as the amino acid involved in the hydrolytic activity of the enzyme. When Y54 was replaced with all other amino acids by site-directed mutagenesis, the hydrolytic activities of the mutated enzymes were drastically altered. The hydrolytic activities of the Y54G, Y54P, Y54T, and Y54W mutated enzymes were remarkably reduced compared with that of the wild-type enzyme, while those of the Y54F and Y54K mutated enzymes were similar to that of the wild-type enzyme. Introducing an amino acid replacement at Y54 also significantly affected the cyclization activity of the amylomaltase. The Y54A, Y54L, Y54R, and Y54S mutated enzymes exhibited cyclization activity that was approximately twofold higher than that of the wild-type enzyme. When the Y54G mutated enzyme was employed for cycloamylose production, the yield of cycloamyloses was more than 90%, and there was no decrease until the end of the reaction.
scite is a Brooklyn-based organization that helps researchers better discover and understand research articles through Smart Citations–citations that display the context of the citation and describe whether the article provides supporting or contrasting evidence. scite is used by students and researchers from around the world and is funded in part by the National Science Foundation and the National Institute on Drug Abuse of the National Institutes of Health.
hi@scite.ai
10624 S. Eastern Ave., Ste. A-614
Henderson, NV 89052, USA
Copyright © 2024 scite LLC. All rights reserved.
Made with 💙 for researchers
Part of the Research Solutions Family.