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排序方式: 共有1341条查询结果,搜索用时 46 毫秒
1.
We present a single-step procedure for the specific mass labeling of unblocked protein N termini. We show that the dye fluorescamine, which is commonly assumed to require mildly alkaline conditions for undergoing a nonspecific reaction with α- and ε-amino groups associated with amino acids, in fact shows a specific reaction only with α-amino groups present at protein N termini when mildly acidic conditions are used. We use this finding to label, identify, and sequence the trypsinolysis-derived N-terminal peptide of lysozyme, using only mass spectrometry, to illustrate how this method could be used with other proteins. 相似文献
2.
The 231-residue capsid (CA) protein of human immunodeficiency virus type 1 (HIV-1) spontaneously self-assembles into tubes with a hexagonal lattice that is believed to mimic the surface lattice of conical capsid cores within intact virions. We report the results of solid-state nuclear magnetic resonance (NMR) measurements on HIV-1 CA tubes that provide new information regarding changes in molecular structure that accompany CA self-assembly, local dynamics within CA tubes, and possible mechanisms for the generation of lattice curvature. This information is contained in site-specific assignments of signals in two- and three-dimensional solid-state NMR spectra, conformation-dependent 15N and 13C NMR chemical shifts, detection of highly dynamic residues under solution NMR conditions, measurements of local variations in transverse spin relaxation rates of amide 1H nuclei, and quantitative measurements of site-specific 15N–15N dipole–dipole couplings. Our data show that most of the CA sequence is conformationally ordered and relatively rigid in tubular assemblies and that structures of the N-terminal domain (NTD) and the C-terminal domain (CTD) observed in solution are largely retained. However, specific segments, including the N-terminal β-hairpin, the cyclophilin A binding loop, the inter-domain linker, segments involved in intermolecular NTD–CTD interactions, and the C-terminal tail, have substantial static or dynamical disorder in tubular assemblies. Other segments, including the 310-helical segment in CTD, undergo clear conformational changes. Structural variations associated with curvature of the CA lattice appear to be localized in the inter-domain linker and intermolecular NTD–CTD interface, while structural variations within NTD hexamers, around local 3-fold symmetry axes, and in CTD–CTD dimerization interfaces are less significant. 相似文献
3.
4.
《Molecular cell》2021,81(24):5099-5111.e8
5.
Prashanth Sirigeri Jois 《Biochemical and biophysical research communications》2010,394(4):1036-1041
Bovine BSP5 belongs to the Binder of SPerm (BSP) family. BSP5 plays a role in the bovine sperm capacitation by promoting cholesterol and phospholipid efflux. The variable N-terminal part in the BSP proteins is the uncharacterized region with no known function. Full-length, N-terminal part, and individual fibronectin type II domains of bovine BSP5 were cloned, expressed and purified from Escherichia coli. His-S tagged N-terminal part showed large variation in migration on SDS-PAGE in comparison to other constructs. Using mass spectrometry it was demonstrated that the His-S-N-terminal part has the expected molecular mass (13 kDa). The recombinant N-terminal part was sensitive to E. coli endogenous proteases during purification. Denaturing purification involving boiling lysis of cells was carried out, as the protein was thermostable. The His-S-N-terminal part lacked structure as determined by CD analysis. Bioinformatics analyses confirmed that the N-terminal part of bovine BSP5 is intrinsically disordered. In addition, bioinformatics analysis indicated that rabbit BSP and multiple forms of BSP proteins of bovine and equine species possess partially or completely disordered N-terminus. The conservation of disorder at the N-terminus in BSP members belonging to different species suggests a role in biological process such as sperm capacitation and/or sperm-egg interactions. 相似文献
6.
Thomas F. Holzman Christine C. Chung Rohinton Edalji David A. Egan Earl J. Gubbins Annemarie Rueter Gail Howard Lana K. Yang Terry M. Pederson Grant A. Krafft et al. 《Journal of Protein Chemistry》1990,9(6):663-672
The gene for human preprorenin was obtained from total RNA prepared from primary human chorion cells. An expression vector was constructed containing an SV40 early promoter, a human preprorenin cDNA, bovine growth hormone poly-A addition signal, and a dihydrofolate reductase (dhfr) expression cassette. This vector was inserted into the DXB-11 Chinese hamster ovary (CHO) cell line. The recombinant protein was exported by CHO cells into the tissue culture media. At harvest the prorenin levels ranged from 1–5 mg/L. For prorenin isolation the cell culture supernatants were processed by filtration, concentration, dialysis, and batch extraction. Preparative-scale isolation of prorenin was accomplished using blue-dye chromatography and size-exclusion chromatography. The isolated prorenin yielded a single SDS-gel band with Mr 40,000. The proprotein was characterized with respect to N-terminal sequence and N-linked sugar composition. Trypsin-activated renin prepared from the proprotein was characterized with respect to N-terminal sequence andpH-activity profile. Enzyme activity was measured with a newly developed fluorogenic peptide substrate containing the P6-P3 sequence of human angiotensinogen. 相似文献
7.
Kunio Yonemasu Takako Sasaki Yoshiko Dohi Charles M. Lapière Betty Nusgens 《生物化学与生物物理学报:疾病的分子基础》1990,1096(1):47-51
C1q, a collagen-like complement protein, was purified from the serum of a ddermatosparactic calf which lacks procollagen N-terminal proteinase (pN-proteinase). The specific hemolytic activity of the serum Clq from the dermatosparactic animal was identical to that of C1q from a normal calf. Gel-filtration of serum from dermatosparactic calf, on Sepharose 6B, showed the presence of C1q-antigenic material at only one position which was identical to the elution position of normal bovine C1q. No differdence, under dissociating conditions, could be seen in the size of the chains of C1q in specific immunoprecipitates isolated from the sera of dermatosparactic and normal animals, as judged by polyacrylamidegel electrophoresis (PAGE) in the presence of sodium dodecyl sulfate (SDS). The C1q from the dermatosparactic animal showed the same N-terminal amino acid and typtic-digest peptide pattern on HPLC as C1q from the normal calf. These results strongly suggest that pN-proteinase is not involved in the extracellular processing of C1q. 相似文献
8.
The N-terminal -amino groups of 1-bungarotoxin (1-Bgt) fromBungarus multicinctus venom were modified with trinitrobenzene sulfonic acid and the modified derivative was separated by high performance liquid chromatography. The trinitrophenylated (TNP) derivative contained two TNP groups at the -amino groups of A chain and B chain and showed a marked decrease in enzymatic activity. Methionine residues at positions 6 and 8 of the A chain were oxidized with chloramine T or cleaved with cyanogen bromide to remove the N-terminal octapeptide. Oxidation of methionine residues and removal of the N-terminal octapeptide caused a precipitous decrease in enzymatic activity, whereas antigenicity remained unchanged. The presence of dihexanoyllecithin influenced the interaction between 1-Bgt and 8-antilinonaphthalene sulfonate (ANS) and revealed that 1-Bgt consists of two types of ANS-binding sites, one at the substrate binding site of the A chain and the other might be at the B chain. The modified derivatives still retained their affinity for Ca2+ and ANS, indicating that the N-terminal region is not involved in Ca2+ and substrate binding. A fluorescence study revealed that the -amino group of the A chain was in the vicinity of substrate binding site and that the TNP -amino groups were in proximity to Trp-19 of the A chain. In addition, the study showed that the N-terminal region is important for stabilizing the architectural environment of Trp-19. The results, together with the proposal that Trp-19 of the A chain is involved in substrate binding, suggest that the N-terminal region of the A chain plays a crucial role in maintaining a functional active site for 1-Bgt. 相似文献
9.
The effect of N-terminal acetylation on the structure of an N-terminal tropomyosin peptide and alpha alpha-tropomyosin. 总被引:1,自引:0,他引:1 下载免费PDF全文
N. J. Greenfield W. F. Stafford S. E. Hitchcock-DeGregori 《Protein science : a publication of the Protein Society》1994,3(3):402-410
We have used a synthetic peptide consisting of the first 30 residues of striated muscle alpha-tropomyosin, with GlyCys added to the C-terminus, to investigate the effect of N-terminal acetylation on the conformation and stability of the N-terminal domain of the coiled-coil protein. In aqueous buffers at low ionic strength, the reduced, unacetylated 32mer had a very low alpha-helical content (approximately 20%) that was only slightly increased by disulfide crosslinking or N-terminal acetylation. Addition of salt (> 1 M) greatly increased the helical content of the peptide. The CD spectrum, the cooperativity of folding of the peptide, and sedimentation equilibrium ultracentrifugation studies showed that it formed a 2-chained coiled coil at high ionic strength. Disulfide crosslinking and N-terminal acetylation both greatly stabilized the coiled-coil alpha-helical conformation in high salt. Addition of ethanol or trifluoroethanol to solutions of the peptide also increased its alpha-helical content. However, the CD spectra and unfolding behavior of the peptide showed no evidence of coiled-coil formation. In the presence of the organic solvents, N-terminal acetylation had very little effect on the conformation or stability of the peptide. Our results indicate that N-terminal acetylation stabilizes coiled-coil formation in the peptide. The effect cannot be explained by interactions with the "helix-dipole" because the stabilization is observed at very high salt concentrations and is independent of pH. In contrast to the results with the peptide, N-terminal acetylation has only small effects on the overall stability of tropomyosin. 相似文献
10.
L. C. Folmar N. D. Denslow † R. A. Wallace ‡ G. LaFleur ‡ T. S. Gross § S. Bonomelli § C. V. Sullivan | 《Journal of fish biology》1995,46(2):255-263