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81.
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Milles S Tyagi S Banterle N Koehler C VanDelinder V Plass T Neal AP Lemke EA 《Journal of the American Chemical Society》2012,134(11):5187-5195
Single-molecule methods have matured into central tools for studies in biology. Foerster resonance energy transfer (FRET) techniques, in particular, have been widely applied to study biomolecular structure and dynamics. The major bottleneck for a facile and general application of these studies arises from the need to label biological samples site-specifically with suitable fluorescent dyes. In this work, we present an optimized strategy combining click chemistry and the genetic encoding of unnatural amino acids (UAAs) to overcome this limitation for proteins. We performed a systematic study with a variety of clickable UAAs and explored their potential for high-resolution single-molecule FRET (smFRET). We determined all parameters that are essential for successful single-molecule studies, such as accessibility of the probes, expression yield of proteins, and quantitative labeling. Our multiparameter fluorescence analysis allowed us to gain new insights into the effects and photophysical properties of fluorescent dyes linked to various UAAs for smFRET measurements. This led us to determine that, from the extended tool set that we now present, genetically encoding propargyllysine has major advantages for state-of-the-art measurements compared to other UAAs. Using this optimized system, we present a biocompatible one-step dual-labeling strategy of the regulatory protein RanBP3 with full labeling position freedom. Our technique allowed us then to determine that the region encompassing two FxFG repeat sequences adopts a disordered but collapsed state. RanBP3 serves here as a prototypical protein that, due to its multiple cysteines, size, and partially disordered structure, is not readily accessible to any of the typical structure determination techniques such as smFRET, NMR, and X-ray crystallography. 相似文献
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84.
Peter Brucker Sigrid Knust Duncan Roper Yakov Zinder 《Mathematical Methods of Operations Research》2000,52(3):369-387
Problems with unit execution time tasks and two identical parallel processors have received a great deal of attention in
scheduling theory. In contrast to the conventional models, where each task requires only one processor, we consider a situation
when a task may require both processors simultaneously. For problems without precedence constraints we present several polynomial
time algorithms which complement recent results of Lee and Cai. We also show that the introduction of precedence constraints
leads to NP-hardness results for maximum lateness and mean flow time objective functions. For the maximum lateness problem,
a family of algorithms, based upon the idea of modified due dates, is considered. The worst case behaviour of these algorithms
is analysed, and it is shown that the same upper bound is tight for each algorithm of this family. 相似文献
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A MRD CI procedure has been used to calculate several electronic states of the hydroperoxyl radical. The basis set is of double-zeta plus polarization quality augmented with s- and p-type bond and Rydberg functions. The vertical excitation energies of the lowest eight doublet and six quartet states are reported. Oscillator strengths for transitions form the ground to upper doublet states were calculated. A cut of the potential energy surfaces along the OOH fragmentation pathway is used to discuss the mechanisms of HO2 photodissociation below 6.4 eV. Arguments are presented which indicate O(1D) rather than O(3P) is the primary dissociation product, and so support the experimental findings rather than theory in the conflict raised earlier on this matter. Ostensibly the dissociation proceeds diabatically on the surface of the initially populated 2A″(1a″ → 2a″) state yielding OH(X2II) + O(1D). 相似文献
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Results of a theoretical study of the Renner-Teller effect in the 1Δg state of methylene are presented. Calculations are based on potential-energy surfaces for 1A1 and 1B1 electronic states obtained by the ab initio MRD CI method. 相似文献