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1.
Srisailam S Kumar TK Rajalingam D Kathir KM Sheu HS Jan FJ Chao PC Yu C 《The Journal of biological chemistry》2003,278(20):17701-17709
Acidic fibroblast growth factor from newt (Notopthalmus viridescens) is a approximately 15-kDa, all beta-sheet protein devoid of disulfide bonds. In the present study, we investigate the effects of 2,2,2-trifluoroethanol (TFE) on the structure of newt acidic fibroblast growth factor (nFGF-1). The protein aggregates maximally in 10% (v/v) TFE. Congo red and thioflavin T binding experiments suggest that the aggregates induced by TFE have properties resembling the amyloid fibrils. Transmission electron microscopy and x-ray fiber diffraction data show that the fibrils (induced by TFE) are straight, unbranched, and have a cross-beta structure with an average diameter of 10-15 A. Preformed fibrils (induced by TFE) of nFGF-1 are observed to seed amyloid-like fibril formation in solutions containing the protein (nFGF-1) in the native beta-barrel conformation. Fluorescence, far-UV CD, anilino-8-napthalene sulfonate binding, multidimensional NMR, and Fourier transformed infrared spectroscopy data reveal that formation of a partially structured intermediate state(s) precedes the onset of the fibrillation process. The native beta-barrel structure of nFGF-1 appears to be disrupted in the partially structured intermediate state(s). The protein in the partially structured intermediate state(s) is found to be "sticky" with a solvent-exposed non-polar surface(s). Amyloid fibril formation appears to occur due to coalescence of the protein in the partially structured intermediate state(s) through solvent-exposed non-polar surfaces and intermolecular beta-sheet formation among the extended, linear beta-strands in the protein. 相似文献
2.
Hsieh HC Kumar TK Chiu CC Yu C 《Biochemical and biophysical research communications》2005,326(1):108-114
Superoxide dismutases (SODs) are important metalloenzymes which protect cells against oxidative stress by scavenging reactive superoxides. Missense mutations in SODs are known to lead to some familial cases of amyotrophic lateral sclerosis and several forms of cancers. In the present study, we investigate the guanidinium hydrochloride (GdnHCl)-induced equilibrium unfolding of apo-manganese superoxide dismutase (apo-MnSOD) isolated from Vibrio alginolyticus using a variety of biophysical techniques. GdnHCl-induced equilibrium unfolding of apo-MnSOD is non-cooperative and involves the accumulation of stable intermediate state(s). Results of 1-anilino-8-naphthalene sulfonate binding experiments suggest that the equilibrium intermediate state(s) accumulates maximally in 1.5M GdnHCl. The intermediate state(s) appears to be obligatory and occurs both in the unfolding and refolding pathways. Size-exclusion chromatography and sedimentation velocity data reveal that the equilibrium intermediate state(s) is multimeric. To our knowledge, this is the first report of the identification of a multimeric intermediate in the unfolding pathway(s) of oligomeric proteins. The formation and dissociation of the multimeric intermediate state(s) appears to dictate the fate of the protein either to refold to its native conformation or misfold and form aggregates as observed in amyotrophic lateral sclerosis. 相似文献
3.
Hui-Chu Hsieh Chi-Cheng Chiu 《Biochemical and biophysical research communications》2004,326(1):108-114
Superoxide dismutases (SODs) are important metalloenzymes which protect cells against oxidative stress by scavenging reactive superoxides. Missense mutations in SODs are known to lead to some familial cases of amyotrophic lateral sclerosis and several forms of cancers. In the present study, we investigate the guanidinium hydrochloride (GdnHCl)-induced equilibrium unfolding of apo-manganese superoxide dismutase (apo-MnSOD) isolated from Vibrio alginolyticus using a variety of biophysical techniques. GdnHCl-induced equilibrium unfolding of apo-MnSOD is non-cooperative and involves the accumulation of stable intermediate state(s). Results of 1-anilino-8-naphthalene sulfonate binding experiments suggest that the equilibrium intermediate state(s) accumulates maximally in 1.5 M GdnHCl. The intermediate state(s) appears to be obligatory and occurs both in the unfolding and refolding pathways. Size-exclusion chromatography and sedimentation velocity data reveal that the equilibrium intermediate state(s) is multimeric. To our knowledge, this is the first report of the identification of a multimeric intermediate in the unfolding pathway(s) of oligomeric proteins. The formation and dissociation of the multimeric intermediate state(s) appears to dictate the fate of the protein either to refold to its native conformation or misfold and form aggregates as observed in amyotrophic lateral sclerosis. 相似文献
4.
Samuel D Kumar TK Srimathi T Hsieh H Yu C 《The Journal of biological chemistry》2000,275(45):34968-34975
The guanidinium hydrochloride (GdnHCl)-induced unfolding of an all beta-sheet protein, the human acidic fibroblast growth factor (hFGF-1), is studied using a variety of biophysical techniques including multidimensional NMR spectroscopy. The unfolding of hFGF-1 in GdnHCl is shown to involve the formation of a stable equilibrium intermediate. Size exclusion chromotagraphy using fast protein liquid chromatography shows that the intermediate accumulates maximally at 0.96 m GdnHCl. 1-Anilinonapthalene 8-sulfonate binding, one-dimensional (1)H NMR, and limited proteolytic digestion experiments suggest that the intermediate has characteristics resembling a molten globule state. Chemical shift perturbation and hydrogen-deuterium exchange monitored by (1)H-(15)N heteronuclear single quantum coherence spectra reveal that profound structural changes in the intermediate state (in 0.96 m GdnHCl) occur in the C-terminal, heparin binding region of the protein molecule. Additionally, results of the stopped flow fluorescence experiments suggest that the kinetic refolding of hFGF-1 proceeds through the accumulation of an intermediate at low concentrations of the denaturant. To our knowledge, the present study is the first report wherein an equilibrium intermediate is characterized in detail in an all beta-barrel protein. 相似文献
5.
Intracellular proteinaceous aggregates (Lewy bodies and Lewy neurites) of alpha-synuclein are hallmarks of neurodegenerative diseases such as Parkinson's disease, dementia with Lewy bodies, and multiple systemic atrophy. However, the molecular mechanisms underlying alpha-synuclein aggregation into such filamentous inclusions remain unknown. An intriguing aspect of this problem is that alpha-synuclein is a natively unfolded protein, with little or no ordered structure under physiological conditions. This raises the question of how an essentially disordered protein is transformed into highly organized fibrils. In the search for an answer to this question, we have investigated the effects of pH and temperature on the structural properties and fibrillation kinetics of human recombinant alpha-synuclein. Either a decrease in pH or an increase in temperature transformed alpha-synuclein into a partially folded conformation. The presence of this intermediate is strongly correlated with the enhanced formation of alpha-synuclein fibrils. We propose a model for the fibrillation of alpha-synuclein in which the first step is the conformational transformation of the natively unfolded protein into the aggregation-competent partially folded intermediate. 相似文献
6.
Identification of rare partially unfolded states in equilibrium with the native conformation in an all beta-barrel protein 总被引:4,自引:0,他引:4
Human acidic fibroblast growth factor 1 (hFGF-1) is an all beta-barrel protein, and the secondary structural elements in the protein include 12 antiparallel beta-strands arranged into a beta-trefoil fold. In the present study, we investigate the stability of hFGF-1 by hydrogen-deuterium exchange as a function of urea concentration. Urea-induced equilibrium unfolding of hFGF-1 monitored by fluorescence and CD spectroscopy suggests that the protein unfolds by a two-state (native to denatured) mechanism. Hydrogen exchange in hFGF-1, under the experimental conditions used, occurs by the EX2 mechanism. In contrast to the equilibrium unfolding events monitored by optical probes, native state hydrogen exchange data show that the beta-trefoil architecture of hFGF-1 does not behave as a single cooperative unit. There are at least two structurally independent units with differing stabilities in hFGF-1. Beta-strands I, II, III, VI, VII, X, XI, and XII fit into the global unfolding isotherm. By contrast, residues in beta-strands IV, V, VIII, and IX exchange by the subfolding isotherm and could be responsible for the occurrence of high-energy partially unfolded state(s) in hFGF-1. There appears to be a broad continuum of stabilities among the four beta-strands (beta-strands IV, V, VIII, and IX) constituting the subglobal folding unit. The slow exchanging residues in hFGF-1 do not represent the folding nucleus of the protein. 相似文献
7.
Dakshinamurthy Rajalingam Charles Loftis Jiashou J. Xu Thallapuranam Krishnaswamy S. Kumar 《Protein science : a publication of the Protein Society》2009,18(5):980-993
Sample preparation for proteomic analysis involves precipitation of protein using 2,2,2‐trichloroacetic acid (TCA). In this study, we examine the mechanism of the TCA‐induced protein precipitation reaction. TCA‐induced protein precipitation curves are U‐shaped and the shape of the curve is observed to be independent of the physicochemical properties of proteins. TCA is significantly less effective in precipitating unfolded states of proteins. Results of the 1‐anilino‐8‐napthalene sulfonate (ANS) and size‐exclusion chromatography, obtained using acidic fibroblast growth factor (aFGF), show that a stable “molten globule‐like” partially structured intermediate accumulates maximally in 5% (w/v) of trichloroacetate. Urea‐induced unfolding and limited proteolytic digestion data reveal that the partially structured intermediate is significantly less stable than the native conformation. 1H‐15N chemical shift perturbation data obtained using NMR spectroscopy indicate that interactions stabilizing the β‐strands at the N‐ and C‐ terminal ends (of aFGF) are disrupted in the trichloroacetate‐induced “MG‐like” state. The results of the study clearly demonstrate that TCA‐induced protein precipitation occurs due to the reversible association of the “MG‐like” partially structured intermediate state(s). In our opinion, the findings of this study provide useful clues toward development of efficient protocols for the isolation and analysis of the entire proteome. 相似文献
8.
G. Jayaraman T.K.S. Kumar T. Sivaraman W.Y. Lin D.K. Chang C. Yu 《International journal of biological macromolecules》1996,18(4):303-306
The thermal unfolding of an all β-sheet protein, cardiotoxin analogue III, from the Taiwan Cobra (Naja naja atra) is studied at pH 2.0, 4.0 and 6.0. At pH 4.0, using circular dichroism and 1-anilino naphthalene-8-sulphonic acid (ANS) fluorescence binding studies, a stable partially structured intermediate is detected at 90°C 相似文献
9.
Srimathi T Kumar TK Chi YH Chiu IM Yu C 《The Journal of biological chemistry》2002,277(49):47507-47516
The structure and dynamics of equilibrium intermediate in the unfolding pathway of the human acidic fibroblast growth factor (hFGF-1) are investigated using a variety of biophysical techniques including multidimensional NMR spectroscopy. Guanidinium hydrochloride (GdnHCl)-induced unfolding of hFGF-1 proceeds with the accumulation of a stable intermediate state. The transition from the intermediate state to the unfolded state(s) is cooperative without the accumulation of additional intermediate(s). The intermediate state induced maximally in 0.96 m GdnHCl is found to be obligatory in the folding/unfolding pathway of hFGF-1. Most of the native tertiary structure interactions are preserved in the intermediate state. (1)H-(15)N chemical shift perturbation data suggest that the residues in the C-terminal segment including those located in the beta-strands IX, X, and XI undergo the most discernible structural change(s) in the intermediate state in 0.96 m GdnHCl. hFGF-1 in the intermediate state (0.96 m GdnHCl) does not bind to its ligand, sucrose octasulfate. Limited proteolytic digestion experiments and hydrogen-deuterium exchange monitored by (15)N heteronuclear single quantum coherence (HSQC) spectra show that the conformational flexibility of the protein in the intermediate state is significantly higher than in the native conformation. (15)N spin relaxation experiments show that many residues located in beta-strands IX, X, and XI exhibit conformational motions in the micro- to millisecond time scale. Analysis of (15)N relaxation data in conjunction with the amide proton exchange kinetics suggests that residues in the beta-strands II, VIII, and XII possibly constitute the stability core of the protein in the near-native intermediate state. 相似文献
10.
Kim YS Randolph TW Manning MC Stevens FJ Carpenter JF 《The Journal of biological chemistry》2003,278(12):10842-10850
Congo red (CR) has been reported to inhibit or enhance amyloid fibril formation by several proteins. To gain insight into the mechanism(s) for these apparently paradoxical effects, we studied as a model amyloidogenic protein, a dimeric immunoglobulin light chain variable domain. With a range of molar ratios of CR, i.e. r = [CR]/[protein dimer], we investigated the aggregation kinetics, conformation, hydrogen-deuterium exchange, and thermal stability of the protein. In addition, we used isothermal titration calorimetry to characterize the thermodynamics of CR binding to the protein. During incubation at 37 degrees C or during thermal scanning, with CR at r = 0.3, 1.3, and 4.8, protein aggregation was greatly accelerated compared with that measured in the absence of the dye. In contrast, with CR at r = 8.8, protein unfolding was favored over aggregation. The aggregates formed with CR at r = 0 or 0.3 were typical amyloid fibrils, but mixtures of amyloid fibrils and amorphous aggregates were formed at r = 1.3 and 4.8. CR decreased the apparent thermal unfolding temperature of the protein. Furthermore, CR perturbed the tertiary structure of the protein without significantly altering its secondary structure. Consistent with this result, CR also increased the rate of hydrogen-deuterium exchange by the protein. Isothermal titration calorimetry showed that CR binding to the protein was enthalpically driven, indicating that binding was mainly the result of electrostatic interactions. Overall, these results demonstrate that at low concentrations, CR binding to the protein favors a structurally perturbed, aggregation-competent species, resulting in acceleration of fibril formation. At high CR concentration, protein unfolding is favored over aggregation, and fibril formation is inhibited. Because low concentrations of CR can promote amyloid fibril formation, the therapeutic utility of this compound or its analogs to inhibit amyloidoses is questionable. 相似文献
11.
Amyloid fibril formation can proceed from different conformations of a partially unfolded protein
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Protein misfolding and aggregation are interconnected processes involved in a wide variety of nonneuropathic, systemic, and neurodegenerative diseases. More generally, if mutations in sequence or changes in environmental conditions lead to partial unfolding of the native state of a protein, it will often aggregate, sometimes into well-defined fibrillar structures. A great deal of interest has been directed at discovering the characteristic features of metastable partially unfolded states that precede the aggregated states of proteins. In this work, human muscle acylphosphatase (AcP) has been first destabilized, by addition of urea or by means of elevated temperatures, and then incubated in the presence of different concentrations of 2,2,2, trifluoroethanol ranging from 5% to 25% (v/v). The results show that AcP is able to form both fibrillar and nonfibrillar aggregates with a high beta-sheet content from partially unfolded states with very different structural features. Moreover, the presence of alpha-helical structure in such a state does not appear to be a fundamental determinant of the ability to aggregate. The lack of ready aggregation under some of the conditions examined here is attributable primarily to the intrinsic properties of the solutions rather than to specific structural features of the partially unfolded states that precede aggregation. Aggregation appears to be favored when the solution conditions promote stable intermolecular interactions, particularly hydrogen bonds. In addition, the structures of the resulting aggregates are largely independent of the conformational properties of their soluble precursors. 相似文献
12.
Liu L Moesner P Kovach NL Bailey R Hamilton AD Sebti SM Harlan JM 《The Journal of biological chemistry》1999,274(47):33334-33340
Integrin-dependent leukocyte adhesion is modulated by alterations in receptor affinity or by post-receptor events. Pretreatment of Jurkat T-cells with the 3-hydroxymethylglutaryl-coenzyme A reductase inhibitor, lovastatin, markedly reduced (IC(50) approximately 1-2 microM) alpha(4)beta(1)-dependent adhesion to fibronectin (FN) stimulated by phorbol 12-myristate 13-acetate (PMA) which modulates post-receptor events. In contrast, lovastatin did not inhibit Jurkat cell adhesion to FN induced by the beta(1) integrin-activating monoclonal antibody (mAb) 8A2, which directly modulates beta(1) integrin affinity. Similarly, pretreatment of U937 cells with lovastatin inhibited PMA-stimulated, but not mAb 8A2-stimulated, alpha(6)beta(1)-dependent leukocyte adhesion to laminin. The inhibition of lovastatin on PMA-stimulated leukocyte adhesion was not mediated by mitogen-activated protein kinase or phosphatidylinositol 3-kinase pathway. The inhibitory effect of lovastatin on PMA-stimulated leukocyte adhesion was reversed by co-incubation with geranylgeraniol, but not with farnesol, with concurrent reversal of the inhibition of protein prenylation as shown by protein RhoA geranylgeranylation. The selective inhibition of protein geranylgeranylation by the specific protein geranylgeranyltransferase-I inhibitor, GGTI-298, blocked PMA-stimulated leukocyte adhesion but not mAb 8A2-induced leukocyte adhesion. The protein farnesyltransferase inhibitor, FTI-277, had no effect on leukocyte adhesion induced by either stimulus. These results demonstrate that protein geranylgeranylation, but not farnesylation, is required for integrin-dependent post-receptor events in leukocyte adhesion. 相似文献
13.
Scarafone N Pain C Fratamico A Gaspard G Yilmaz N Filée P Galleni M Matagne A Dumoulin M 《PloS one》2012,7(3):e31253
Nine neurodegenerative disorders, called polyglutamine (polyQ) diseases, are characterized by the formation of intranuclear amyloid-like aggregates by nine proteins containing a polyQ tract above a threshold length. These insoluble aggregates and/or some of their soluble precursors are thought to play a role in the pathogenesis. The mechanism by which polyQ expansions trigger the aggregation of the relevant proteins remains, however, unclear. In this work, polyQ tracts of different lengths were inserted into a solvent-exposed loop of the β-lactamase BlaP and the effects of these insertions on the properties of BlaP were investigated by a range of biophysical techniques. The insertion of up to 79 glutamines does not modify the structure of BlaP; it does, however, significantly destabilize the enzyme. The extent of destabilization is largely independent of the polyQ length, allowing us to study independently the effects intrinsic to the polyQ length and those related to the structural integrity of BlaP on the aggregating properties of the chimeras. Only chimeras with 55Q and 79Q readily form amyloid-like fibrils; therefore, similarly to the proteins associated with diseases, there is a threshold number of glutamines above which the chimeras aggregate into amyloid-like fibrils. Most importantly, the chimera containing 79Q forms amyloid-like fibrils at the same rate whether BlaP is folded or not, whereas the 55Q chimera aggregates into amyloid-like fibrils only if BlaP is unfolded. The threshold value for amyloid-like fibril formation depends, therefore, on the structural integrity of the β-lactamase moiety and thus on the steric and/or conformational constraints applied to the polyQ tract. These constraints have, however, no significant effect on the propensity of the 79Q tract to trigger fibril formation. These results suggest that the influence of the protein context on the aggregating properties of polyQ disease-associated proteins could be negligible when the latter contain particularly long polyQ tracts. 相似文献
14.
In highly purified tonoplast fractions from Acer pseudoplatanus cells, the in vitro reversible phosphorylation of proteins affected only a restricted set of polypeptides. The phosphorylation process has been shown to be dramatically stimulated by calcium via the mediation of calmodulin as the transducer. The protein kinase(s) was totally inhibited by micromolar concentrations of a calmodulin antagonist. Tonoplast appears to be potentially a good experimental system for the evaluation of the effects of protein phosphorylation on membrane properties in plants.Abbreviations CaM
calmodulin
- EGTA
ethylene glycol-bis-(-amino ethylether)N,N,NN-tetraacetate
- SDS-PAGE
sodium dodecylsulphate polyacrylamide gel electrophoresis
- TCA
trichloroacetic acid 相似文献
15.
Dislocation of an endoplasmic reticulum membrane glycoprotein involves the formation of partially dislocated ubiquitinated polypeptides 总被引:2,自引:0,他引:2
Accumulation of improperly folded polypeptides in the endoplasmic reticulum (ER) can trigger a stress response that leads to the export of aberrant proteins into the cytosol and their ultimate proteasomal degradation. Human cytomegalovirus encodes a type I glycoprotein, US11, that binds to nascent MHC class I heavy chain molecules and causes their dislocation from the ER to the cytosol where they are degraded by the proteasome. Examination of US11-mediated class I degradation has identified a host of cellular proteins involved in the dislocation reaction, including the cytosolic AAA ATPase p97, the membrane protein Derlin-1, and the E3 ubiquitin ligase Sel1L. However, the intermediate steps occurring between the initiation of dislocation and full extraction of the misfolded substrate into the cytosol are not known. We demonstrate that US11 itself undergoes ER export and proteasomal degradation and utilize this system to define multiple steps of US11 dislocation. Treatment of US11-expressing cells with proteasome inhibitor resulted in the accumulation of glycosylated and ubiquitinated species as well as a deglycosylated US11 intermediate. Subcellular fractionation of proteasome-inhibited US11 cells demonstrated that deglycosylated intermediates continued to be integrated within the ER membrane, suggesting that the proteasome functions in the latter steps of dislocation. The data supports a model in which US11 is modified with ubiquitin, whereas the transmembrane region is integrated in the ER membrane, and deglycosylation occurs before complete dislocation. 相似文献
16.
Newt fibroblast growth factor (nFGF-1) is an approximately 15-kDa all beta-sheet protein devoid of disulfide bonds. Urea-induced equilibrium unfolding of nFGF-1, monitored by steady state fluorescence and far-UV circular dichroism spectroscopy, is cooperative with no detectable intermediate(s). Urea-induced unfolding of nFGF-1 is reversible, but the percentage of the protein recovered in the native state depends on the time of incubation of the protein in the denaturant. The yield of the protein in the native state decreases with the increase in time of incubation in the denaturant. The failure of the protein to refold to its native state is not due to trivial chemical reactions that could possibly occur upon prolonged incubation in the denaturant. (1)H-(15)N heteronuclear single quantum coherence (HSQC) spectra, limited proteolytic digestion, and fluorescence data suggest that the misfolded state(s) of nFGF-1 has structural features resembling that of the denatured state(s). GroEL, in the presence of ATP, is observed to rescue the protein from being trapped in the misfolded state(s). (1)H-(15)N HSQC data of nFGF-1, acquired in the denatured state(s) (in 8 m urea), suggest that the protein undergoes subtle time-dependent structural changes in the denaturant. To our knowledge, this report for the first time demonstrates that the commitment to adapt unproductive pathways leading to protein misfolding/aggregation occurs in the denatured state ensemble. 相似文献
17.
Trinucleotide expansions leading to an extended poly-L-alanine segment in the poly (A) binding protein PABPN1 cause fibril formation
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Scheuermann T Schulz B Blume A Wahle E Rudolph R Schwarz E 《Protein science : a publication of the Protein Society》2003,12(12):2685-2692
The nuclear poly(A) binding protein (PABPN1) stimulates poly(A) polymerase and controls the lengths of poly(A) tails during pre-mRNA processing. The wild-type protein possesses 10 consecutive Ala residues immediately after the start methionine. Trinucleotide expansions in the coding sequence result in an extension of the Ala stretch to maximal 17 Ala residues in total. Individuals carrying the trinucleotide expansions suffer from oculopharyngeal muscular dystrophy (OPMD). Intranuclear inclusions consisting predominantly of PABPN1 have been recognized as a pathological hallmark of the genetic disorder. To elucidate the molecular events that lead to disease, recombinant PABPN1, and N-terminal fragments of the protein with varying poly-L-alanine stretches were analyzed. As the full-length protein displayed a strong tendency to aggregate into amorphous deposits, soluble N-terminal fragments were also studied. Expansion of the poly-L-alanine sequence to the maximal length observed in OPMD patients led to an increase of alpha-helical structure. Upon prolonged incubation the protein was found in fibrils that showed all characteristics of amyloid-like fibers. The lag-phase of fibril formation could be reduced by seeding. Structural analysis of the fibrils indicated antiparallel beta-sheets. 相似文献
18.
High-affinity binding of the staphylococcal HarA protein to haptoglobin and hemoglobin involves a domain with an antiparallel eight-stranded beta-barrel fold
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Dryla A Hoffmann B Gelbmann D Giefing C Hanner M Meinke A Anderson AS Koppensteiner W Konrat R von Gabain A Nagy E 《Journal of bacteriology》2007,189(1):254-264
Iron scavenging from the host is essential for the growth of pathogenic bacteria. In this study, we further characterized two staphylococcal cell wall proteins previously shown to bind hemoproteins. HarA and IsdB harbor homologous ligand binding domains, the so called NEAT domain (for "near transporter") present in several surface proteins of gram-positive pathogens. Surface plasmon resonance measurements using glutathione S-transferase (GST)-tagged HarAD1, one of the ligand binding domains of HarA, and GST-tagged full-length IsdB proteins confirmed high-affinity binding to hemoglobin and haptoglobin-hemoglobin complexes with equilibrium dissociation constants (K(D)) of 5 to 50 nM. Haptoglobin binding could be detected only with HarA and was in the low micromolar range. In order to determine the fold of this evolutionarily conserved ligand binding domain, the untagged HarAD1 protein was subjected to nuclear magnetic resonance spectroscopy, which revealed an eight-stranded, purely antiparallel beta-barrel with the strand order (-beta1 -beta2 -beta3 -beta6 -beta5 -beta4 -beta7 -beta8), forming two Greek key motifs. Based on structural-homology searches, the topology of the HarAD1 domain resembles that of the immunoglobulin (Ig) fold family, whose members are involved in protein-protein interactions, but with distinct structural features. Therefore, we consider that the HarAD1/NEAT domain fold is a novel variant of the Ig fold that has not yet been observed in other proteins. 相似文献
19.
We have investigated the possible covalent binding of intermediates in prostaglandin (PG) biosynthesis to tissue macromolecules. Following incubation of arachidonic acid -1-[14]C (AA) with guinea pig lung microsomes, radioactivity was associated with the microsomal protein which was not dissociated from the protein by exhaustive solvent extraction. Furthermore, filtration of the protein complex through a Sephadex G-25 column failed to dissociate the radioactivity from the protein. This probably indicates covalent binding of AA metabolite(s) to protein. [3]H-PGE2, [3]H-PGF2α, and [3]H-thromboxane B2 (TXB2) did not show this high affinity binding to microsomal protein. The covalent binding of AA metabolites was greatly reduced in denatured microsomes and was inhibited by the addition of glutathione (GSH) or indomethacin to the incubation mixtures. Chromatographic analysis of the water layers obtained from microsomal incubations with either [3]H-AA or [3]H-GSH suggested the presence of one or more glutathione conjugates derived from AA. These studies indicate that most likely an intermediate formed during PG synthesis from AA covalently binds to tissue macromolecules. This covalent binding may be of physiological and pathological significance. 相似文献
20.
The association of amyloid P-component (AP) with the amyloid fibril: an updated method for amyloid fibril protein isolation 总被引:6,自引:0,他引:6
An amyloid fibril isolation procedure is proposed which uses citrate as well as saline washes to dissociate the calcium dependent linkage of amyloid P-component (AP) from the amyloid fibril. In two amyloid rich tissues, the amount of AP was quantitated in each saline and citrate wash and totalled 13.8% and 20.8% of the amyloid fibrils isolated. The amount of AP removed from these and 22 additional amyloid rich tissues was greater than had previously been recognized. AP protein was present in tissue only when amyloid fibrils were present. It could not be found in normal non-amyloidotic tissue, nor could it be found in tissue sediment after the fibrils were removed. 相似文献