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Dr. Sandra Liebscher Dr. Sebastian Mathea Dr. Tobias Aumüller Dr. Andreas Pech Prof. Dr. Frank Bordusa 《Chembiochem : a European journal of chemical biology》2021,22(7):1201-1204
Fluorescent fusion proteins are powerful tools for studying biological processes in living cells, but universal application is limited due to the voluminous size of those tags, which might have an impact on the folding, localization or even the biological function of the target protein. The designed biocatalyst trypsiligase enables site-directed linkage of small-sized fluorescence dyes on the N terminus of integral target proteins located in the outer membrane of living cells through a stable native peptide bond. The function of the approach was tested by using the examples of covalent derivatization of the transmembrane proteins CD147 as well as the EGF receptor, both presented on human HeLa cells. Specific trypsiligase recognition of the site of linkage was mediated by the dipeptide sequence Arg-His added to the proteins’ native N termini, pointing outside the cell membrane. The labeling procedure takes only about 5 minutes, as demonstrated for couplings of the fluorescence dye tetramethyl rhodamine and the affinity label biotin as well. 相似文献
3.
Dr. Adarshi P. Welegedara Dr. Ansis Maleckis Ruchira Bandara Dr. Mithun C. Mahawaththa Iresha Dilhani Herath Yi Jiun Tan Dr. Angeliki Giannoulis Prof. Daniella Goldfarb Prof. Gottfried Otting Prof. Thomas Huber 《Chembiochem : a European journal of chemical biology》2021,22(8):1480-1486
The selenol group of selenocysteine is much more nucleophilic than the thiol group of cysteine. Selenocysteine residues in proteins thus offer reactive points for rapid post-translational modification. Herein, we show that selenoproteins can be expressed in high yield and purity by cell-free protein synthesis by global substitution of cysteine by selenocysteine. Complete alkylation of solvent-exposed selenocysteine residues was achieved in 10 minutes with 4-chloromethylene dipicolinic acid (4Cl-MDPA) under conditions that left cysteine residues unchanged even after overnight incubation. GdIII−GdIII distances measured by double electron–electron resonance (DEER) experiments of maltose binding protein (MBP) containing two selenocysteine residues tagged with 4Cl-MDPA-GdIII were indistinguishable from GdIII−GdIII distances measured of MBP containing cysteine reacted with 4Br-MDPA tags. 相似文献
4.
A heavy crude oil has been treated with deuterated alkylating reagents (CD3I and C2D5I) and directly analyzed without any prior fractionation and chromatographic separation by high-field Orbitrap Fourier Transform Mass Spectrometry (FTMS) and Fourier Transform Ion Cyclotron Resonance Mass Spectrometry (FT-ICR MS) using electrospray ionization (ESI). The reaction of a polycyclic aromatic sulfur heterocycles (PASHs) dibenzothiophene (DBT), in the presence of silver tetrafluoroborate (AgBF4) with ethyl iodide (C2H5I) in anhydrous dichloroethane (DCE) was optimized as a sample reaction to study heavy crude oil mixtures, and the reaction yield was monitored and determined by proton nuclear magnetic resonance spectroscopy (1H-NMR). The obtained conditions were then applied to a mixture of standard aromatic CH-, N-, O- and S-containing compounds and then a heavy crude oil, and only sulfur-containing compounds were selectively alkylated. The deuterium labeled alkylating reagents, iodomethane-d3 (CD3I) and iodoethane-d5 (C2D5I), were employed to the alkylation of heavy crude oil to selectively differentiate the tagged sulfur species from the original crude oil. 相似文献
5.
The classical overlapping Schwarz algorithm is here extended to the spectral element discretization of linear elastic problems, for both homogeneous and heterogeneous compressible materials. The algorithm solves iteratively the resulting preconditioned system of linear equations by the conjugate gradient or GMRES methods. The overlapping Schwarz preconditioned technique is then applied to the numerical approximation of elastic waves with spectral elements methods in space and implicit Newmark time advancing schemes. The results of several numerical experiments, for both elastostatic and elastodynamic problems, show that the convergence rate of the proposed preconditioning algorithm is independent of the number of spectral elements (scalability), is independent of the spectral degree in case of generous overlap, otherwise it depends inversely on the overlap size. Some results on the convergence properties of the spectral element approximation combined with Newmark schemes for elastic waves are also presented. 相似文献
6.
7.
H. Hoteit Ph. Ackerer R. Mos J. Erhel B. Philippe 《International journal for numerical methods in engineering》2004,61(14):2566-2593
In this paper, we introduce an extension of Van Leer's slope limiter for two‐dimensional discontinuous Galerkin (DG) methods on arbitrary unstructured quadrangular or triangular grids. The aim is to construct a non‐oscillatory shock capturing DG method for the approximation of hyperbolic conservative laws without adding excessive numerical dispersion. Unlike some splitting techniques that are limited to linear approximations on rectangular grids, in this work, the solution is approximated by means of piecewise quadratic functions. The main idea of this new reconstructing and limiting technique follows a well‐known approach where local maximum principle regions are defined by enforcing some constraints on the reconstruction of the solution. Numerical comparisons with some existing slope limiters on structured as well as on unstructured meshes show a superior accuracy of our proposed slope limiters. Copyright © 2004 John Wiley & Sons, Ltd. 相似文献
8.
D. Roy 《International journal for numerical methods in engineering》2004,61(5):764-790
Sample pathwise numerical integration of noise-driven engineering dynamical systems cannot generally be performed beyond a limited level of accuracy, especially when the noise processes are modelled using (filtered) white noises. Recently, a locally transversal linearization (LTL) strategy has been proposed by the author (Proc Roy Soc London A 2001; 457 :539–566) for direct integration of deterministic and stochastic non-linear dynamical systems. The present effort is focussed on a host of extensions along with detailed theoretical error analyses of the linearization approach, especially as applied to problems in non-linear stochastic engineering dynamics. Thus, to begin with, estimates of local and global error orders in the basic LTL scheme are obtained separately for the displacement and velocity vectors when the system is driven either by a set of additive noises or by an arbitrary combination of (independently evolving) additive and multiplicative noises. Following this, a new family of higher-order LTL schemes is proposed in order to improve upon the basic LTL method and the associated error orders are established. A stepwise implementation of the lower- and higher-order versions of the LTL method, along with certain computational aspects, is also outlined. The proposed schemes are numerically illustrated, to a limited extent, for a single degree-of-freedom (SDOF) and a two degree-of-freedom (TDOF) non-linear engineering systems under additive and/or multiplicative white noise excitations. Copyright © 2004 John Wiley & Sons, Ltd. 相似文献
9.
In this work a new class of numerical methods for the BGK model of kinetic equations is presented. In principle, schemes of
any order of accuracy in both space and time can be constructed with this technique. The methods proposed are based on an
explicit–implicit time discretization. In particular the convective terms are treated explicitly, while the source terms are
implicit. In this fashion even problems with infinite stiffness can be integrated with relatively large time steps. The conservation
properties of the schemes are investigated. Numerical results are shown for schemes of order 1, 2 and 5 in space, and up to
third-order accurate in time. 相似文献
10.
We consider an integrated scheduling and distribution model in which jobs completed by two different machines must be bundled together for delivery. The objective is to minimize the sum of the delivery cost and customers' waiting costs. Such a model not only attempts to coordinate the job schedules on both machines, but also aims to coordinate the machine schedules with the delivery plan. Polynomial-time heuristics and approximation schemes are developed for the model with only direct shipments as well as the general model with milk-run deliveries. 相似文献