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161.
162.
Valérie Copié John A. Battles John M. Schwab Dennis A. Torchia 《Journal of biomolecular NMR》1996,7(4):335-340
Summary Nearly complete backbone 1H, 15N and 13C signal assignments are reported for -hydroxydecanoyl thiol ester dehydrase, a 39-kDa homodimer containing 342 amino acids. Although 15N relaxation data show that the protein has a rotational correlation time of 18 ns, assignments were derived from triple-resonance experiments recorded at 500 MHz and pH 6.8, without deuteration. The Chemical Shift Index, CSI, identified two long helices and numerous -strands in dehydrase. The CSI predictions are in close agreement with the secondary structure identified in the recently derived crystal structure, particularly when one takes account of the numerous bulges in the -strands. The assignment of dehydrase and a large deuterated protein [Yamazaki et al. (1994) J. Am. Chem. Soc., 116, 11655–11666] suggest that assignment of 40–60 kDa proteins is feasible. Hence, further progress in understanding the chemical shift/structure relationship could open the way to determine the structures of such large proteins.
Supplementary Material is available on request, comprising Table S1 listing the spectral parameters; Table S2 listing the assignments; Fig. S1 showing the 2D 1H–15N HSQC spectrum; Fig. S2 showing sequential NOEs, secondary shifts, H-exchange and 3JHN
data; and Fig. S3 showing plots of the H, C, CO and C Chemical Shift Indexes.To whom correspondence should be addressed. 相似文献
163.
Sjaak Peelen Sybren S. Wijmenga Paul J. A. Erbel Robert L. Robson Robert R. Eady Jacques Vervoort 《Journal of biomolecular NMR》1996,7(4):315-330
Summary The 1H, 15N and 13C backbone and 1H and 13C beta resonance assignments of the long-chain flavodoxin from Azotobacter chroococcum (the 20-kDa nifF product, flavodoxin-2) in its oxidized form were made at pH 6.5 and 30°C using heteronuclear multidimensional NMR spectroscopy. Analysis of the NOE connectivities, together with amide exchange rates, 3JHnH coupling constants and secondary chemical shifts, provided extensive solution secondary structure information. The secondary structure consists of a five-stranded parallel -sheet and five -helices. One of the outer regions of the -sheet shows no regular extended conformation, whereas the outer strand 4/6 is interrupted by a loop, which is typically observed in long-chain flavodoxins. Two of the five -helices are nonregular at the N-terminus of the helix. Loop regions close to the FMN are identified. Negatively charged amino acid residues are found to be mainly clustered around the FMN, whereas a cluster of positively charged residues is located in one of the -helices. Titration of the flavodoxin with the Fe protein of the A. chroococcum nitrogenase enzyme complex revealed that residues Asn11, Ser68 and Asn72 are involved in complex formation between the flavodoxin and Fe protein. The interaction between the flavodoxin and the Fe protein is influenced by MgADP and is of electrostatic nature.Abbreviations SQ
semiquinone
- FMN
riboflavin 5-monophosphate; nif, nitrogen fixation
- TSP
3-(trimethylsilyl)propionate sodium salt
- DSS
2,2-dimethyl-2-silapentane-5-sulfonate sodium salt
Supplementary Material is available on request, comprising a Materials and Methods section for the expression and purification of the A. chroococcum flavodoxin, a Table S1 containing the parameters of the titration of A. chroococcum flavodoxin with the Fe protein, and a Table S2 containing the 15N, HN, 13C, 1H, 13C, 1H and 13CO chemical shifts.To whom correspondence should be addressed. 相似文献
164.
Summary Sequence-specific 1H and 15N resonance assignments have been made for 137 of the 146 nonprolyl residues in oxidized Desulfovibrio desulfuricans [Essex 6] flavodoxin. Assignments were obtained by a concerted analysis of the heteronuclear three-dimensional 1H-15N NOESY-HMQC and TOCSY-HMQC data sets, recorded on uniformly 15N-enriched protein at 300 K. Numerous side-chain resonances have been partially or fully assigned. Residues with overlapping 1HN chemical shifts were resolved by a three-dimensional 1H-15N HMQC-NOESY-HMQC spectrum. Medium-and long-range NOEs, 3JNH
coupling constants, and 1HN exchange data indicate a secondary structure consisting of five parallel -strands and four -helices with a topology similar to that of Desulfovibrio vulgaris [Hidenborough] flavodoxin. Prolines at positions 106 and 134, which are not conserved in D. vulgaris flavodoxin, contort the two C-terminal -helices.Abbreviations CSI
chemical shift index
- DQF-COSY
double-quantum-filtered correlation spectroscopy
- DIPSI
decoupling in the presence of scalar interactions
- FMN
flavin mononucleotide
- GARP
globally optimized alternating phase rectangular pulse
- HMQC
heteronuclear multiple-quantum coherence
- HSQC
heteronuclear single-quantum coherence
- NOE
nuclear Overhauser effect
- NOESY
nuclear Overhauser enhancement spectroscopy
- TOCSY
total correlation spectroscopy
- TPPI
time-proportional phase increments
- TSP
3-(trimethylsilyl)propionic-2,2,3,3-d
4 acid, sodium salt 相似文献
165.
Yu-Sen Wang Anne F. Frederick Mary M. Senior Barbara A. Lyons Stuart Black Paul Kirschmeier Louise M. Perkins Oswald Wilson 《Journal of biomolecular NMR》1996,7(2):89-98
Summary The growth factor receptor-bound protein-2 (Grb2) is an adaptor protein that mediates signal transduction pathways. Chemical shift assignments were obtained for the SH2 domain of Grb2 by heteronuclear NMR spectroscopy, employing the uniformly 13C-/15N-enriched protein as well as the protein containing selectively 15N-enriched amino acids. Using the Chemical Shift Index (CSI) method, the chemical shift indices of four nuclei, 1H, 13C, 13C and 13CO, were used to derive the secondary structure of the protein. Nuclear Overhauser enhancements (NOEs) were then employed to confirm the secondary structure. The CSI results were compared to the secondary structural elements predicted for the Grb2 SH2 domain from a sequence alignment [Lee et al. (1994) Structure, 2, 423–438]. The core structure of the SH2 domain contains an antiparallel -sheet and two -helices. In general, the secondary structural elements determined from the CSI method agree well with those predicted from the sequence alignment.Abbreviations crk
viral p47gag-crk
- EGF
epidermal growth factor
- GAP
GTPase-activating protein
- PI3K
phosphatidylinositol-3-kinase
- PLC-
phospholipase-C-, shc, src homologous and collagen
- src
sarcoma family of nonreceptor tyrosine kinase 相似文献
166.
报道了一种修饰SOD的方法。所得硬脂酸修饰SOD比活力为每毫克蛋白10000单位,经鉴定已达均一程度。测得其分子量为35000.修饰SOD和天然SOD在紫外光区的最大吸收均在265nm。修饰SOD对温度、pH、蛋白酶水解的稳定性比天然SOD增强,且免疫原性消除。在低浓度的某些有机介质中活性比在水中高。 相似文献
167.
毛细管气相色谱/质谱/计算机联用仪器分析结果表明,在四川省成都市一人工种植的银木(Cinnamomumseptentrionale)种群中,其枝叶精油主要化学组成在各植株间存在很大差异,发现精油存在1,8-桉叶油素,樟脑,异丁香酚甲醇和9-氧代橙花叔醇等四个化学类型.除异丁香酚甲醚类型外,其余类型均为第一次报道.综观樟属其它种的化学类型研究可见,化学类型在樟属植物中普遍存在种内多型性和种间共性. 相似文献
168.
用化学修饰、内源荧光和荧光淬灭等方法研究了油麻藤凝集素(MSL)的溶液构象变化和微环境的构象特征。研究发现MSL分子中总共有9个色氨酸(Trp)残基,它们的荧光能被丙烯酰胺淬灭,但不易为KI接近而淬灭,MSL经N-溴代琥珀酰亚胺(NBS)修饰后,其内源性荧光发射谱发生相应变化,结果表明MSL分子中部分Trp残基埋藏于分子内部,而位于分子表面的Trp残基可能处于分子的疏水袋中。 相似文献
169.
用自制的氨基PEG化试剂rIL-2进行化学修饰,研究了试剂浓度,溶液pH,反应时间等与PEca-rIL-2产率及IL-2活性保持之间的关系,建立了一套获得稳定修饰度的PEG-rIL-2的方法。研究发现,反应时间跟修饰度关系不大;溶液pH对修饰度有一定的影响,中性pH以上反应都可进行;而试剂浓度直接决定修饰度的高低,过量越多,修饰度越高,而生物活性保留也越低;但低度修饰,对活性几乎没有影响,可保留活性在95%左右。 相似文献
170.
水稻巯基蛋白酶抑制剂(CPI)经用二硫苏糖醇,对氯汞苯甲酸和碘乙酸修饰后,对木瓜蛋白酶的抑制活性并无改变;用N-乙基顺丁烯二酰亚胺与CPI反应,可以测出CPI分子内有19个巯基被修饰,被修饰后,抑制活性仍无改变,表明水稻CPI的抑制活性不需要巯基参与;应用N-溴代丁二酰亚胺与CPI反应,可测出CPI分子内有2个Trp被修饰,修饰后,抑制活性全部丧失,表明Trp是保持抑制活性所必需的基团。水稻巯基蛋白酶抑制剂和丝氨酸蛋白酶抑制剂对稻瘟病菌丝体的生长均有抑制作用,但后者的抑制作用比前者更强,若将两种抑制剂混合使用,则对稻瘟病菌丝体的抑制作用非常强烈;当抑制剂加入量达72μg时,即可产生明显的抑制作用。 相似文献