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981.
Probing minimal independent folding units in dihydrofolate reductase by molecular dissection. 总被引:7,自引:5,他引:2 下载免费PDF全文
C. V. Gegg K. E. Bowers C. R. Matthews 《Protein science : a publication of the Protein Society》1997,6(9):1885-1892
Molecular dissection was employed to identify minimal independent folding units in dihydrofolate reductase (DHFR) from Escherichia coli. Eight overlapping fragments of DHFR, spanning the entire sequence and ranging in size from 36 to 123 amino acids, were constructed by chemical cleavage. These fragments were designed to examine the effect of tethering multiple elements of secondary structure on folding and to test if the secondary structural domains represent autonomous folding units. CD and fluorescence spectroscopy demonstrated that six fragments containing up to a total of seven alpha-helices or beta-strands and, in three cases, the adenine binding domain (residues 37-86), are largely disordered. A stoichiometric mixture of the two fragments comprising the large discontinuous domain, 1-36 and 87-159, also showed no evidence for folding beyond that observed for the isolated fragments. A fragment containing residues 1-107 appears to have secondary and tertiary structure; however, spontaneous self-association made it impossible to determine if this structure solely reflects the behavior of the monomeric form. In contrast, a monomeric fragment spanning residues 37-159 possesses significant secondary and tertiary structure. The urea-induced unfolding of fragment 37-159 in the presence of 0.5 M ammonium sulfate was found to be a well-defined, two-state process. The observation that fragment 37-159 can adopt a stable native fold with unique, aromatic side-chain packing is quite striking because residues 1-36 form an integral part of the structural core of the full-length protein. 相似文献
982.
Importance of the release of strand 1C to the polymerization mechanism of inhibitory serpins. 总被引:4,自引:2,他引:2 下载免费PDF全文
W. S. Chang J. Whisstock P. C. Hopkins A. M. Lesk R. W. Carrell M. R. Wardell 《Protein science : a publication of the Protein Society》1997,6(1):89-98
Serpin polymerization is the underlying cause of several diseases, including thromboembolism, emphysema, liver cirrhosis, and angioedema. Understanding the structure of the polymers and the mechanism of polymerization is necessary to support rational design of therapeutic agents. Here we show that polymerization of antithrombin is sensitive to the addition of synthetic peptides that interact with the structure. A 12-m34 peptide (homologous to P14-P3 of antithrombin reactive loop), representing the entire length of s4A, prevented polymerization totally. A 6-mer peptide (homologous to P14-P9 of antithrombin) not only allowed polymerization to occur, but induced it. This effect could be blocked by the addition of a 5-mer peptide with s1C sequence of antithrombin or by an unrelated peptide representing residues 26-31 of cholecystokinin. The s1C or cholecystokinin peptide alone was unable to form a complex with native antithrombin. Moreover, an active antitrypsin double mutant, Pro 361-->Cys, Ser 283-->Cys, was engineered for the purpose of forming a disulfide bond between s1C and s2C to prevent movement of s1C. This mutant was resistant to polymerization if the disulfide bridge was intact, but, under reducing conditions, it regained the potential to polymerize. We have also modeled long-chain serpin polymers with acceptable stereochemistry using two previously proposed loop-A-sheet and loop-C-sheet polymerization mechanisms and have shown both to be sterically feasible, as are "mixed" linear polymers. We therefore conclude that the release of strand 1C must be an element of the mechanism of serpin polymerization. 相似文献
983.
S. Gillis B. C. Furie B. Furie H. Patel M. C. Huberty M. Switzer W. B. Foster H. A. Scoble M. D. Bond 《Protein science : a publication of the Protein Society》1997,6(1):185-196
The gamma-carboxyglutamic acid (Gla) domains of the vitamin K-dependent blood coagulation proteins contain 10 highly conserved Gla residues within the first 33 residues, but factor IX is unique in possessing 2 additional Gla residues at positions 36 and 40. To determine their importance, factor IX species lacking these Gla residues were isolated from heterologously expressed human factor IX. Using ion-exchange chromatography, peptide mapping, mass spectrometry, and N-terminal sequencing, we have purified and identified two partially carboxylated recombinant factor IX species; factor IX/gamma 40E is uncarboxylated at residue 40 and factor IX/gamma 36,40E is uncarboxylated at both residues 36 and 40. These species were compared with the fully gamma-carboxylated recombinant factor IX, unfractionated recombinant factor IX, and plasma-derived factor IX. As monitored by anti-factor IX:Ca (II)-specific antibodies and by the quenching of intrinsic fluorescence, all these factor IX species underwent the Ca(II)-induced conformational transition required for phospholipid membrane binding and bound equivalently to phospholipid vesicles composed of phosphatidylserine, phosphatidylcholine, and phosphatidylethanolamine. Endothelial cell binding was also similar in all species, with half-maximal inhibition of the binding of 125I-labeled plasma-derived factor IX at concentrations of 2-6 nM. Functionally, factor IX/gamma 36,40E and factor IX/gamma 40E were similar to fully gamma-carboxylated recombinant factor IX and plasma-derived factor IX in their coagulant activity and in their ability to participate in the activation of factor X in the tenase complex both with synthetic phospholipid vesicles and activated platelets. However, Gla 36 and Gla 40 represent part of the epitope targeted by anti-factor IX:Mg(II)-specific antibodies because these antibodies bound factor IX preferentially to factor IX/gamma 36,40E and factor IX/gamma 40E. These results demonstrate that the gamma-carboxylation of glutamic acid residues 36 and 40 in human factor IX is not required for any function of factor IX examined. 相似文献
984.
Crystallization and preliminary crystallographic analysis of recombinant human P38 MAP kinase. 下载免费PDF全文
S. Pav D. M. White S. Rogers K. M. Crane C. L. Cywin W. Davidson J. Hopkins M. L. Brown C. A. Pargellis L. Tong 《Protein science : a publication of the Protein Society》1997,6(1):242-245
The recombinant human p38 MAP kinase has been expressed and purified from both Escherichia coli and SF9 cells, and has been crystallized in two forms by the hanging drop vapor diffusion method using PEG as precipitant. Both crystal forms belong to space group P2(1)2(1)2(1). The cell parameters for crystal form 1 are a = 65.2 A, b = 74.6 A and c = 78.1 A. Those for crystal form 2 are a = 58.3 A, b = 68.3 A and c = 87.9 A. Diffraction data to 2.0 A resolution have been collected on both forms. 相似文献
985.
S. H. Park R. T. Raines 《Protein science : a publication of the Protein Society》1997,6(11):2344-2349
Green fluorescent protein (GFP) is autofluorescent. This property has made GFP useful in monitoring in vivo activities such as gene expression and protein localization. We find that GFP can be used in vitro to reveal and characterize protein-protein interactions. The interaction between the S-peptide and S-protein fragments of ribonuclease A was chosen as a model system. GFP-tagged S-peptide was produced, and the interaction of this fusion protein with S-protein was analyzed by two distinct methods: fluorescence gel retardation and fluorescence polarization. The fluorescence gel retardation assay is a rapid method to demonstrate the existence of a protein-protein interaction and to estimate the dissociation constant (Kd) of the resulting complex. The fluorescence polarization assay is an accurate method to evaluate Kd in a specified homogeneous solution and can be adapted for the high-throughput screening of protein or peptide libraries. These two methods are powerful new tools to probe protein-protein interactions. 相似文献
986.
Direct sequence analysis of proteins by in-source fragmentation during delayed ion extraction. 总被引:1,自引:1,他引:0 下载免费PDF全文
J. J. Lennon K. A. Walsh 《Protein science : a publication of the Protein Society》1997,6(11):2446-2453
Continuous segments of amino acid sequence information as long as 41 residues have been deduced by interpretation of matrix-assisted laser desorption/ionization-generated ion signals dominated by Cn fragmentation within the ion source of a linear time-of-flight mass spectrometer utilizing delayed ion extraction. The technique has been applied successively to five proteins of mass 12.2 kDa to 18.3 kDa, yielding segments of continuous sequence as long as 41 residues without the need for prior proteolytic fragmentation. Intact crosslinks such as disulfides or heme linkages interrupt the generation of these data. 相似文献
987.
Structural heterogeneity of the various forms of apomyoglobin: implications for protein folding. 总被引:1,自引:1,他引:0 下载免费PDF全文
R. Gilmanshin R. B. Dyer R. H. Callender 《Protein science : a publication of the Protein Society》1997,6(10):2134-2142
Temperature-induced denaturation transitions of different structural forms of apomyoglobin were studied monitoring intrinsic tryptophan fluorescence. It was found that the tryptophans are effectively screened from solvent both in native and acid forms throughout most of the temperature range tested. Thus, the tryptophans' surrounding do not show a considerable change in structure where major protein conformational transitions have been found in apomyoglobin using other techniques. At high temperatures and under strong destabilizing conditions, the tryptophans' fluorescence parameters show sigmoidal thermal denaturation. These results, combined with previous studies, show that the structure of this protein is heterogeneous, including native-like (tightly packed) and molten globule-like substructures that exhibit conformation (denaturation) transitions under different conditions of pH and temperature (and denaturants). The results suggest that the folding of this protein proceeds via two "nucleation" events whereby native-like contacts are formed. One of these events, which involves AGH "core" formation, appears to occur very early in the folding process, even before significant hydrophobic collapse in the rest of the protein molecule. From the current studies and other results, a rather detailed picture of the folding of myoglobin is presented, on the level of specific structures and their thermodynamical properties as well as formation kinetics. 相似文献
988.
Conformation of P22 tailspike folding and aggregation intermediates probed by monoclonal antibodies. 总被引:3,自引:2,他引:1 下载免费PDF全文
M. A. Speed T. Morshead D. I. Wang J. King 《Protein science : a publication of the Protein Society》1997,6(1):99-108
The partitioning of partially folded polypeptide chains between correctly folded native states and off-pathway inclusion bodies is a critical reaction in biotechnology. Multimeric partially folded intermediates, representing early stages of the aggregation pathway for the P22 tailspike protein, have been trapped in the cold and isolated by nondenaturing polyacrylamide gel electrophoresis (PAGE) (speed MA, Wang DIC, King J. 1995. Protein Sci 4:900-908). Monoclonal antibodies against tailspike chains discriminate between folding intermediates and native states (Friguet B, Djavadi-Ohaniance L, King J, Goldberg ME. 1994. J Biol Chem 269:15945-15949). Here we describe a nondenaturing Western blot procedure to probe the conformation of productive folding intermediates and off-pathway aggregation intermediates. The aggregation intermediates displayed epitopes in common with productive folding intermediates but were not recognized by antibodies against native epitopes. The nonnative epitope on the folding and aggregation intermediates was located on the partially folded N-terminus, indicating that the N-terminus remained accessible and nonnative in the aggregated state. Antibodies against native epitopes blocked folding, but the monoclonal directed against the N-terminal epitope did not, indicating that the conformation of the N-terminus is not a key determinant of the productive folding and chain association pathway. 相似文献
989.
Statistical significance of hierarchical multi-body potentials based on Delaunay tessellation and their application in sequence-structure alignment. 总被引:2,自引:1,他引:1 下载免费PDF全文
P. J. Munson R. K. Singh 《Protein science : a publication of the Protein Society》1997,6(7):1467-1481
Statistical potentials based on pairwise interactions between C alpha atoms are commonly used in protein threading/fold-recognition attempts. Inclusion of higher order interaction is a possible means of improving the specificity of these potentials. Delaunay tessellation of the C alpha-atom representation of protein structure has been suggested as a means of defining multi-body interactions. A large number of parameters are required to define all four-body interactions of 20 amino acid types (20(4) = 160,000). Assuming that residue order within a four-body contact is irrelevant reduces this to a manageable 8,855 parameters, using a nonredundant dataset of 608 protein structures. Three lines of evidence support the significance and utility of the four-body potential for sequence-structure matching. First, compared to the four-body model, all lower-order interaction models (three-body, two-body, one-body) are found statistically inadequate to explain the frequency distribution of residue contacts. Second, coherent patterns of interaction are seen in a graphic presentation of the four-body potential. Many patterns have plausible biophysical explanations and are consistent across sets of residues sharing certain properties (e.g., size, hydrophobicity, or charge). Third, the utility of the multi-body potential is tested on a test set of 12 same-length pairs of proteins of known structure for two protocols: Sequence-recognizes-structure, where a query sequence is threaded (without gap) through the native and a non-native structure; and structure-recognizes-sequence, where a query structure is threaded by its native and another non-native sequence. Using cross-validated training, protein sequences correctly recognized their native structure in all 24 cases. Conversely, structures recognized the native sequence in 23 of 24 cases. Further, the score differences between correct and decoy structures increased significantly using the three- or four-body potential compared to potentials of lower order. 相似文献
990.
Determination of the binding frame of the chaperone SecB within the physiological ligand oligopeptide-binding protein. 总被引:5,自引:3,他引:2 下载免费PDF全文
V. F. Smith S. J. Hardy L. L. Randall 《Protein science : a publication of the Protein Society》1997,6(8):1746-1755
Chaperone proteins demonstrate the paradoxical ability to bind ligands rapidly and with high affinity but with no apparent sequence specificity. To learn more about this singular property, we have mapped the binding frame of the chaperone SecB from E. coli on the oligopeptide-binding protein. Similar studies performed on the maltose-binding and galactose-binding proteins revealed centrally positioned binding frames of approximately 160 aminoacyl residues. The work described here shows that OppA, which is significantly longer than the previously studied ligands, has a binding frame that covers 460 amino acids, nearly the entire length of the protein. We propose modes of binding to account for the data. 相似文献