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The free energy profiles for the chemical reaction of the guanosine triphosphate hydrolysis GTP + H2O → GDP + Pi by Ras‐GAP for the wild‐type and G13V mutated Ras were computed by using molecular dynamics protocols with the QM(ab initio)/MM potentials. The results are consistent with the recent measurements of reaction kinetics in Ras‐GAP showing about two‐order reduction of the rate constant upon G13V mutation in Ras: the computed activation barrier on the free energy profile is increased by 3 kcal/mol upon the G13V replacement. The major reason for a higher energy barrier is a shift of the “arginine finger” (R789 from GAP) from the favorable position in the active site. The results of simulations provide support for the mechanism of the reference reaction according to which the Q61 side chain directly participates in chemical transformations at the proton transfer stage. Proteins 2015; 83:1046–1053. © 2015 Wiley Periodicals, Inc.  相似文献   
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倪郁  宋超  王小清 《生态学报》2014,34(15):4160-4166
以野生型拟南芥与蜡质突变体cer1、cer4为试验材料,通过研究核盘菌胁迫对拟南芥茎表皮蜡质结构及组分含量的影响,揭示核盘菌侵染与表皮蜡质的关系。扫描电镜结果显示,野生型拟南芥蜡质晶体以垂直于表面的杆状、块状结构为主;突变体cer1晶体类型以水平的松针状、块状结构为主;突变体cer4蜡质晶体以垂直片层结构为主。核盘菌胁迫下,拟南芥蜡质晶体结构及分布形态发生变化。蜡质层结构在核盘菌胁迫下表现为:杆状、松针状蜡质晶体减少—蜡质晶体熔融—表皮"囊状凸起"—表皮膜层破裂。这些结构变化有利于病菌突破角质层屏障而侵入到植株体内。色质谱分析结果显示:与野生型相比,cer1突变体烷、次级醇、酮类显著减少;cer4突变体表现为一级醇含量减少。接种核盘菌后,野生型拟南芥与蜡质突变体一级醇类显著增加(cer1增加不显著);烷类、次级醇类、酮类含量与蜡质总量均显著减少,表明蜡质前体物质在受到核盘菌胁迫后更多地通过酰基还原途径生成一级醇,从而减少了由脱羰基途径所生成的蜡质组分。核盘菌通过改变表皮蜡质晶体结构与化学组分分泌量来促进侵染。  相似文献   
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In the X-ray structure of the staphylococcal nuclease–Ca2+ ?3′,5′-pdTp complex, the conformation of the inhibitor 3′,5′-pdTp is distroteed Lys-70* and Lys-71* from an adjacent molecule of staphylococcal nuclease (Loll, P.J., Lattman, E.E. Proteins 5 : 183-201, 1989). In order to correct this crystal packing problem, the solution conformation of enzyme-bound 3′,5′-pdTp in the staphylococcal nuclease–metal–pdTp Complex determined by NMR methods was docked into the X-ray structure of the enzyme [Weber, D. J., Serpersu, E. H., Gittis, A. G., Lattman, E. E., Mildvan, A. S. (preceding paper)]. In the NMR-docked structure, the 5′-phophate of 3′,5′-pdTp overlaps with that in the X-ray Structure. However the 3′-phosphate accepts a hydrogen bond from Lys-49 (2.89Å) rather than from Lys-84 (8.63 Å), and N3 of thymine donates a hydrogen bond to the OH of Tyr-115 (3.16 Å) which does not occur in the X-ray structure (5.28 Å). These interactions have been tested by binding studies of 3′,5′-pdTp, Ca2+, and Mn2+ to the K49A, K84A, and Y115A mutants of staphylococcal nuclease using water proton relaxation rate and EPR methods. Each mutant was fully active and structurally intact, as found by CD and two-dimensional NMR spectroscopy, but bound Ca2+ 9.1- to 9.9-fold more weakly than the wild-type enzyme. While thye K84A mutation did not significantly weaken 3′,5′-pdTp binding to the enzyme (1.5 ± 0.7 fold), the K49A mutation weakened 3′,5′-pdTp binding to the enzyme by the factor of 4.4 ± 1.8-fold. Similarly, the Y115A mutation weakened 3′,5′-pdTp binding to the enzyme 3.6 ± 1.6-fold. Comparable weakening effects of these mutations were found on the binding of Ca2+-3′,5′-pdTp. These results are more readily explained by the NMR-docked structure of staphylococcal nuclease-metal-3′,5′-pdTp than by the X-ray structure. © 1993 Wiley-Liss, Inc.  相似文献   
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Twenty-eight site-directed mutations were introduced into the fission yeast gene (pcn1 +) that encodes proliferating cell nuclear antigen (PCNA) and their in vivo effects analyzed in a strain with a null pcn1 background. Mutants defective in enhancing processivity of DNA polymerase δ have previously been identified. In this study, we assessed all of the mutants for their sensitivities to temperature, hydroxyurea, UV irradiation and methyl methanesulfonate (MMS), and specific mutants were also tested for sensitivity to γ irradiation. One cold-sensitive allele, pcn1-3, was characterized in detail. This mutant had a recessive cold-sensitive cdc phenotype and showed sensitivity to hydroxyurea, UV, and γ irradiation. At the non-permissive temperature pcn1-3 protein was able to form homotrimers in solution and showed increased stimulation of both synthetic activity and processivity of DNA polymerase δ relative to the wild-type Pcn1+ protein. Epistasis analyses indicated that pcn1-3 is defective in the repair pathway involving rad2 + but not defective in the classical nucleotide excision repair pathway involving rad13 + . Furthermore, pcn1-3 is either synthetically or conditionally lethal in null checkpoint rad backgrounds and displays a mitotic catastrophe phenotype in these backgrounds. A model for how pcn1-3 defects may affect DNA repair and replication is presented. Received: 5 July 1997 / Accepted: 10 October 1997  相似文献   
7.
Flowering response and plant form of photomorphogenic mutants (hy1, hy2, hy3, hy4 and hy5) of Arabidopsis thaliana (L.), a long-day plant, were examined in long and short days. There were only slight differences among genotypes including Landsberg wild type with respect to the flowering time under long days. The effect of 1 h light-(night)-breaks of far-red, red, blue and white light given in the middle of the dark period of plants grown under short days, was studied. Effects of far-red light applied at the end or the beginning of the main photoperiod on flowering and plant form were also examined. The light-breaks with all the above mentioned light qualities promoted floral initiation of all the genotypes including the wild type in terms of both the flowering time and the number of rosette leaves. In general, far-red light was most effective. It is possible to classify the hy-mutants into 3 groups by their responses to light-breaks under short day conditions: (a) Mutants hy2 and hy3, which have a reduced number of rosette leaves, and flower early. Red light is as effective as far-red light. The wavelength of light-breaks is relatively unimportant for flowering response. (b) Mutants hy4, hy5 and Landsberg wild type, which have a greater number of rosette leaves, and flower relatively late. The effectiveness of light-breaks is in the following order, far-red, blue, and red light, which is in reverse order to the transformation of phytochrome to the Pfr form. (c) Mutant hy1, which behaves anomalously with respect to relations between flowering time and number of rosette leaves; late flowering with reduced number of rosette leaves. Red, blue and far-red light are effective, but white light is ineffective for reducing the number of rosette leaves. When far-red light was given in the middle of the night or at the end of the main photoperiod, it markedly reduced the number of rosette leaves compared to those grown under short days for all the genotypes, while when applied at the beginning of the main photoperiod far-red light did not affect the number of rosette leaves. Different effects on the plant form dependent on the time of treatment with far-red light-breaks are also discussed.  相似文献   
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A single cell of Listeria monocytogenes attached on food contact surfaces can be a potential source of cross-contamination in a food-processing plant. To see whether internalin A (InlA) and B (InlB), major surface proteins on L. monocytogenes, play a significant role in the attachment process, wild-type L. monocytogenes EGD (LM_EGD) and its isogenic internalin-negative mutants (LM_EGDΔinlA, LM_EGDΔinlB, and LM_EGDΔinlAB) were used to determine attachment strength on inert glass surface. Western blot analysis using InlA and InlB antibodies confirmed the absence of InlA in LM_EGDΔinlA, InlB in LM_EGDΔinlB, and both InlA and InlB in LM_EGDΔinlAB. Regardless of initial attachment numbers, LM_EGD which expressed both InlA and InlB proteins exhibited the strongest attachment strength while the double mutant (LM_EGDΔinlAB) exhibited the weakest. The two single mutants (LM_EGDΔinlA and LM_EGDΔinlB) that expressed only one type of the internalins were shown to have intermediate attachment strength. These results suggest that both InlA and InlB expression play a significant role in the attachment strength of L. monocytogenes on glass surface.  相似文献   
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Terrestrial plants produce extracellular aliphatic biopolyesters that modify cell walls of specific tissues. Epidermal cells synthesize cutin, a polyester of glycerol and modified fatty acids that constitutes the framework of the cuticle that covers aerial plant surfaces. Suberin is a related lipid polyester that is deposited on the cell walls of certain tissues, including the root endodermis and the periderm of tubers, tree bark and roots. These lipid polymers are highly variable in composition among plant species, and often differ among tissues within a single species. Here, we describe a detailed protocol to study the monomer composition of cutin in Arabidopsis thaliana leaves by sodium methoxide (NaOMe)-catalyzed depolymerisation, derivatization, and subsequent gas chromatography-mass spectrometry (GC/MS) analysis. This method can be used to investigate the monomers of insoluble polyesters isolated from whole delipidated plant tissues bearing either cutin or suberin. The method can by applied not only to characterize the composition of lipid polymers in species not previously analyzed, but also as an analytical tool in forward and reverse genetic approaches to assess candidate gene function.  相似文献   
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