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1.
T Langer  C Lu  H Echols  J Flanagan  M K Hayer  F U Hartl 《Nature》1992,356(6371):683-689
The main stress proteins of Escherichia coli function in an ordered protein-folding reaction. DnaK (heat-shock protein 70) recognizes the folding polypeptide as an extended chain and cooperates with DnaJ in stabilizing an intermediate conformational state lacking ordered tertiary structure. Dependent on GrpE and ATP hydrolysis, the protein is then transferred to GroEL (heat-shock protein 60) which acts catalytically in the production of the native state. This sequential mechanism of chaperone action may represent an important pathway for the folding of newly synthesized polypeptides.  相似文献   

2.
Trigger factor and DnaK cooperate in folding of newly synthesized proteins.   总被引:22,自引:0,他引:22  
The role of molecular chaperones in assisting the folding of newly synthesized proteins in the cytosol is poorly understood. In Escherichia coli, GroEL assists folding of only a minority of proteins and the Hsp70 homologue DnaK is not essential for protein folding or cell viability at intermediate growth temperatures. The major protein associated with nascent polypeptides is ribosome-bound trigger factor, which displays chaperone and prolyl isomerase activities in vitro. Here we show that delta tig::kan mutants lacking trigger factor have no defects in growth or protein folding. However, combined delta tig::kan and delta dnaK mutations cause synthetic lethality. Depletion of DnaK in the delta tig::kan mutant results in massive aggregation of cytosolic proteins. In delta tig::kan cells, an increased amount of newly synthesized proteins associated transiently with DnaK. These findings show in vivo activity for a ribosome-associated chaperone, trigger factor, in general protein folding, and functional cooperation of this protein with a cytosolic Hsp70. Trigger factor and DnaK cooperate to promote proper folding of a variety of E. coli proteins, but neither is essential for folding and viability at intermediate growth temperatures.  相似文献   

3.
Three-dimensional NMR spectroscopy of a protein in solution   总被引:3,自引:0,他引:3  
The geometric information used to solve three-dimensional (3D) structures of proteins by NMR spectroscopy resides in short (less than 5 A) interproton-distance data. To obtain these distances, the 1H-NMR spectrum must first be assigned using correlation and nuclear Overhauser effect (NOE) experiments to demonstrate through-bond (scalar) and through-space connectivities, respectively. Because the NOE is proportional to r-6, distance information can then be derived. The increased resolution afforded by extending NMR experiments into a second dimension enables one to detect and interpret effects that would not be possible in one dimension owing to extensive spectral overlap and much reduced information. A number of small protein structures have previously been solved in this way. Extending this methodology to larger proteins, however, requires yet an additional improvement in resolution as overlap of cross-peaks in the two-dimensional (2D) NMR spectra present a major barrier to their unambiguous identification. One way of increasing the resolution is to extend the 2D-NMR experiments into a third dimension. We report here the applicability of three-dimensional NMR to macromolecules using the 46-residue protein alpha 1-purothionin as an example.  相似文献   

4.
NMR analysis of a 900K GroEL GroES complex   总被引:16,自引:0,他引:16  
Fiaux J  Bertelsen EB  Horwich AL  Wüthrich K 《Nature》2002,418(6894):207-211
Biomacromolecular structures with a relative molecular mass (M(r)) of 50,000 to 100,000 (50K 100K) have been generally considered to be inaccessible to analysis by solution NMR spectroscopy. Here we report spectra recorded from bacterial chaperonin complexes ten times this size limit (up to M(r) 900K) using the techniques of transverse relaxation-optimized spectroscopy and cross-correlated relaxation-enhanced polarization transfer. These techniques prevent deterioration of the NMR spectra by the rapid transverse relaxation of the magnetization to which large, slowly tumbling molecules are otherwise subject. We tested the resolving power of these techniques by examining the isotope-labelled homoheptameric co-chaperonin GroES (M(r) 72K), either free in solution or in complex with the homotetradecameric chaperonin GroEL (M(r) 800K) or with the single-ring GroEL variant SR1 (M(r) 400K). Most amino acids of GroES show the same resonances whether free in solution or in complex with chaperonin; however, residues 17 32 show large chemical shift changes on binding. These amino acids belong to a mobile loop region of GroES that forms contacts with GroEL. This establishes the utility of these techniques for solution NMR studies that should permit the exploration of structure, dynamics and interactions in large macromolecular complexes.  相似文献   

5.
G J Phillips  T J Silhavy 《Nature》1990,344(6269):882-884
The use of lacZ gene fusions, producing a hybrid protein containing an amino terminus specified by a target gene fused to the functional carboxy terminus of beta-galactosidase, has facilitated the study of protein targeting in various organisms. One of the best characterized fusions in Escherichia coli is phi(lamB-lacZ)42-1(Hyb), which produces a hybrid protein with the signal sequence and 181 N-terminal amino acids of the exported protein LamB, attached to LacZ. In common with other LacZ hybrids, the LamB-LacZ(42-1) protein is poorly exported from E. coli, conferring a Lac+ phenotype. beta-Galactosidase activity decreases markedly when cells producing the LamB-LacZ protein are grown at 42 degrees C or when a heat-shock response is induced at lower temperatures by overproducing heat-shock factor RpoH3, indicating the LacZ hybrids are being efficiently targeted to the cell envelope. We now report that the heat-shock proteins DnaK and GroEL can, in sufficient amounts, decrease beta-galactosidase activity and facilitate the export of lacZ-hybrid proteins.  相似文献   

6.
BindingSitesofLa3+onCamodulin*FengYuping(冯玉萍),YuanHaodan(袁浩丹)+,ZhouYuxiang(周玉祥)+,NingYongcheng(宁永成),ZhangRiqing(张日清)+Departme...  相似文献   

7.
E P Nikonowicz  A Pardi 《Nature》1992,355(6356):184-186
Multidimensional heteronuclear NMR has revolutionized solution structure determinations of proteins. But this technique has not been applied to nucleic acids because of difficulties in the synthesis of isotopically (13C and/or 15N) labelled molecules. Here we report the application of three-dimensional heteronuclear NMR to the study of a uniformly 13C/15N or 15N-labelled RNA duplex of defined sequence. These experiments simplify resonance assignment and the analysis of proton-proton nuclear Overhauser effects (and therefore distance information) in the molecule. Our results show that it is now possible to determine the structures of larger and more complex RNAs using multidimensional heteronuclear NMR.  相似文献   

8.
(Mg-ATP)-dependent self-assembly of molecular chaperone GroEL   总被引:7,自引:0,他引:7  
The important Escherichia coli heat-shock protein GroEL of relative molecular mass 57,259 is a typical molecular chaperone. It possesses ATPase activity and interacts in ATP-driven reactions with non-folded proteins to stimulate their correct folding and/or assembly by preventing the formation of improper protein structures or aggregates. As GroEL is isolated and functions as a 20-25S tetradecameric particle (GroELp), the question arises--what is the mechanism of its own assembly? Here we show the (Mg-ATP)-dependent self-stimulation ('self-chaperoning') in vitro of GroELp reassembly from its monomeric state.  相似文献   

9.
As the co-chaperone of DnaK/Hsp70 protein, DnaJ/Hsp40 protein influences the synthesis and assembly of the protein complex by regulating ATPase activity of DnaK/Hsp70 protein. By employing the modified method of cDNA representational difference analysis, a homologous fragment of DnaJ was isolated from the deregulated carrot somatic embryos, and it was further used as the probe to screen the cDNA library of carrot somatic embryo deregulated for 12 h. As the result, DcJ1 gene, the homologous gene of DnaJ, was isolated from carrot. Sequence analysis showed that its coding region is 1257 bp, which codes 418 amino acids and comprises 3 highly-conserved characteristic domains. Southern blot analysis suggested that the DcJ1 gene seems to be a single copy in the genome, while Northern blot result indicated that DcJ1 expresses only in roots and its degree of expression changes obviously with the regulation-deregulation process. These results suggest that DcJ1 is correlated with the early development of carrot somatic embryo radicle.  相似文献   

10.
11.
本文给出了利用51VNMR研究鸡卵铁传递蛋白的实验过程和结果,显示出卵铁传递蛋白的两个结合金属的部位(C末端与N末端)在结构和作用上的相似性和差别,并表明了51VNMR是钒与卵铁传递蛋白结合的高灵敏度的探测工具,可用于研究钒与其它金属蛋白的结合。  相似文献   

12.
The fast reaction of the actin-based cytoskeleton in motile cells after stimulation with a chemoattractant requires a signal-transduction chain that creates a very specific environment at distinct regions beneath the plasma membrane. Dictyostelium hisactophilin, a unique actin-binding protein, is a submembranous pH sensor that signals slight changes of the H+ concentration to actin by inducing actin polymerization and binding to microfilaments only at pH values below seven. It has a relative molecular mass of 13.5K and its most unusual feature is the presence of 31 histidine residues among its total of 118 amino acids. The transduction of an external signal from the plasma membrane to the cytoskeleton is poorly understood. Here we report the protein's structure in solution determined by nuclear magnetic resonance spectroscopy. The nuclear Overhauser effect intensities of the three-dimensional nuclear Overhauser spectra were used directly in the calculations. The overall folding of histactophilin is similar to that of interleukin-1 beta and fibroblast growth factor, but the primary amino-acid sequence of hisactophilin is unrelated to these two proteins.  相似文献   

13.
Identification of in vivo substrates of the chaperonin GroEL   总被引:22,自引:0,他引:22  
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14.
The Arc repressor, which is involved in the switch between lysis and lysogeny of Salmonella bacteriophage P22, does not belong to any of the known classes of DNA-binding proteins. Mutagenesis studies show that the DNA-binding region is located in the 15 N-terminal amino-acid residues. We have now determined the three-dimensional structure of the Arc dimer from an extensive set of interproton-distance data obtained from 1H NMR spectroscopy. A priori, intra- and inter-monomer nuclear Overhauser effects (NOEs) cannot be distinguished for a symmetric dimer. But by using the homology with the Escherichia coli Met repressor we could interpret the NOEs unambiguously in an iterative structure refinement procedure. The final structure satisfies a large set of NOE constraints (1,352 for the dimer). It shows a strongly intertwined dimer, in which residues 8-14 of different monomers form an antiparallel beta-sheet. A model for the Arc repressor-operator complex can account for all available biochemical and genetic data. In this model two Arc dimers bind with their beta-sheet regions in successive major grooves on one side of the DNA helix, similar to the Met repressor interaction. Thus, Arc and Met repressors are members of the same family of proteins, which contain an antiparallel beta-sheet as the DNA-binding motif.  相似文献   

15.
A number of functionally important actions of proteins are mediated by short, intrinsically disordered peptide segments, but the molecular interactions that allow disordered domains to mediate their effects remain a topic of active investigation. Many K+ channel proteins, after initial channel opening, show a time-dependent reduction in current flux, termed 'inactivation', which involves movement of mobile cytosolic peptide segments (approximately 20-30 residues) into a position that physically occludes ion permeation. Peptide segments that produce inactivation show little amino-acid identity and tolerate appreciable mutational substitutions without disrupting the inactivation process. Solution nuclear magnetic resonance of several isolated inactivation domains reveals substantial conformational heterogeneity with only minimal tendency to ordered structures. Channel inactivation mechanisms may therefore help us to decipher how intrinsically disordered regions mediate functional effects. Whereas many aspects of inactivation of voltage-dependent K+ channels (Kv) can be described by a simple one-step occlusion mechanism, inactivation of the voltage-dependent large-conductance Ca2+-gated K+ (BK) channel mediated by peptide segments of auxiliary β-subunits involves two distinguishable kinetic steps. Here we show that two-step inactivation mediated by an intrinsically disordered BK β-subunit peptide involves a stereospecific binding interaction that precedes blockade. In contrast, blocking mediated by a Shaker Kv inactivation peptide is consistent with direct, simple occlusion by a hydrophobic segment without substantial steric requirement. The results indicate that two distinct types of molecular interaction between disordered peptide segments and their binding sites produce qualitatively similar functions.  相似文献   

16.
Sugase K  Dyson HJ  Wright PE 《Nature》2007,447(7147):1021-1025
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17.
利用核磁共振测定木材润胀细胞壁的水分含量与孔径分布   总被引:1,自引:0,他引:1  
【目的】利用核磁共振冻融分析技术测定杉木与杨木两种速生材细胞壁润胀状态吸着水含量与孔隙分布情况,为改性剂粒径选择与改性效果评价提供方法。【方法】常温下获得试样内水分T2弛豫信号总量,通过核磁冻融分析系统对试样进行降温处理,检测不同冷冻温度条件试样内未冻结水分信号量; 依据Gibbs-Thomson效应确定凝固点降低与孔径的关系,并以此分析孔径分布。【结果】核磁冻融法测定杉木和杨木吸着水饱和含量约为38%,高于通过吸湿外推法估算数值,与溶剂排出法、多孔板法、离心法结果相近; 核磁冻融分析法避免了常温条件按照T2弛豫分布确定吸着水含量偏小的现象; 两种速生材试样细胞壁润胀状态孔径小于1.59 nm的孔隙占比约为75%,大于4.56 nm的孔隙占比不超过6%,与溶剂排出法、光谱标记法结果相符。【结论】核磁共振冻融分析技术可较为便捷准确地获得木材吸着水含量与细胞壁孔隙分布,其结果可用于指导改性剂粒径的选择与改性效果评价。  相似文献   

18.
Mori T  Vale RD  Tomishige M 《Nature》2007,450(7170):750-754
Kinesin-1 (conventional kinesin) is a dimeric motor protein that carries cellular cargoes along microtubules by hydrolysing ATP and moving processively in 8-nm steps. The mechanism of processive motility involves the hand-over-hand motion of the two motor domains ('heads'), a process driven by a conformational change in the neck-linker domain of kinesin. However, the 'waiting conformation' of kinesin between steps remains controversial-some models propose that kinesin adopts a one-head-bound intermediate, whereas others suggest that both the kinesin heads are bound to adjacent tubulin subunits. Addressing this question has proved challenging, in part because of a lack of tools to measure structural states of the kinesin dimer as it moves along a microtubule. Here we develop two different single-molecule fluorescence resonance energy transfer (smFRET) sensors to detect whether kinesin is bound to its microtubule track by one or two heads. Our FRET results indicate that, while moving in the presence of saturating ATP, kinesin spends most of its time bound to the microtubule with both heads. However, when nucleotide binding becomes rate-limiting at low ATP concentrations, kinesin waits for ATP in a one-head-bound state and makes brief transitions to a two-head-bound intermediate as it walks along the microtubule. On the basis of these results, we suggest a model for how transitions in the ATPase cycle position the two kinesin heads and drive their hand-over-hand motion.  相似文献   

19.
把束缚核子内海克从物理上分为两部分:一部分是伴随价夸克受核束缚效应影响的云海夸克,另一部分是能摆脱核子的要票才而逃逸到核环境中的背景海夸克。首先,给出了核束缚效应对核内束缚核子中价夸克、云海夸克及相应胶子的纵向动量几率分布函数的最低级修正形式;借助核动量守恒条件定出了背景海夸克及相应胶子的动量几率分布函数,根据部分子内禀横向动量分布与动量分布间的制约关系及我们关于核环境中部分子间相互演化的观点,建  相似文献   

20.
Global unfolding of a substrate protein by the Hsp100 chaperone ClpA.   总被引:18,自引:0,他引:18  
The bacterial protein CIpA, a member of the Hsp100 chaperone family, forms hexameric rings that bind to the free ends of the double-ring serine protease ClpP. ClpA directs the ATP-dependent degradation of substrate proteins bearing specific sequences, much as the 19S ATPase 'cap' of eukaryotic proteasomes functions in the degradation of ubiquitinated proteins. In isolation, ClpA and its relative ClpX can mediate the disassembly of oligomeric proteins; another similar eukaryotic protein, Hsp104, can dissociate low-order aggregates. ClpA has been proposed to destabilize protein structure, allowing passage of proteolysis substrates through a central channel into the ClpP proteolytic cylinder. Here we test the action of ClpA on a stable monomeric protein, the green fluorescent protein GFP, onto which has been added an 11-amino-acid carboxy-terminal recognition peptide, which is responsible for recruiting truncated proteins to ClpAP for degradation. Fluorescence studies both with and without a 'trap' version of the chaperonin GroEL, which binds non-native forms of GFP, and hydrogen-exchange experiments directly demonstrate that ClpA can unfold stable, native proteins in the presence of ATP.  相似文献   

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