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1.
Application of fourth derivative absorption spectroscopy to protein quantitation during purification
A method for protein quantitation in the presence of nonprotein cellular components is described. The method is based on measurement of two tryptophan-specific signals in the fourth derivative of the protein's ultraviolet absorption spectrum, a peak at 283 nm and a trough at 288 nm. The amplitude between these two extremes is shown to vary linearly with protein concentration for bovine serum albumin and the outer membrane vesicles of Neissera meningitidis even when these protein solutions are supplemented with enough nucleic acid to completely obscure the parent absorption spectrum of the protein. The utility of this method as an in-process assay during isolation of a protein is demonstrated by comparing estimates of protein content from fourth derivative spectroscopy with those from the Lowry assay for samples at several steps along the isolation pathway for outer membrane vesicles of N. meningitidis. The advantages and limitations of the present method are discussed. 相似文献
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3.
UNC-11, a Caenorhabditis elegans AP180 Homologue, Regulates the Size and Protein Composition of Synaptic Vesicles
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Michael L. Nonet Andrea M. Holgado Faraha Brewer Craig J. Serpe Betty A. Norbeck Julianne Holleran Liping Wei Erika Hartwieg Erik M. Jorgensen Aixa Alfonso 《Molecular biology of the cell》1999,10(7):2343-2360
The unc-11 gene of Caenorhabditis elegans encodes multiple isoforms of a protein homologous to the mammalian brain-specific clathrin-adaptor protein AP180. The UNC-11 protein is expressed at high levels in the nervous system and at lower levels in other tissues. In neurons, UNC-11 is enriched at presynaptic terminals but is also present in cell bodies. unc-11 mutants are defective in two aspects of synaptic vesicle biogenesis. First, the SNARE protein synaptobrevin is mislocalized, no longer being exclusively localized to synaptic vesicles. The reduction of synaptobrevin at synaptic vesicles is the probable cause of the reduced neurotransmitter release observed in these mutants. Second, unc-11 mutants accumulate large vesicles at synapses. We propose that the UNC-11 protein mediates two functions during synaptic vesicle biogenesis: it recruits synaptobrevin to synaptic vesicle membranes and it regulates the size of the budded vesicle during clathrin coat assembly. 相似文献
4.
Luo X Schwarz-Linek U Botting CH Hensel R Siebers B White MF 《Journal of bacteriology》2007,189(2):403-409
The genomes of the related crenarchaea Pyrobaculum aerophilum and Thermoproteus tenax lack any obvious gene encoding a single-stranded DNA binding protein (SSB). SSBs are essential for DNA replication, recombination, and repair and are found in all other genomes across the three domains of life. These two archaeal genomes also have only one identifiable gene encoding a chromatin protein (the Alba protein), while most other archaea have at least two different abundant chromatin proteins. We performed a biochemical screen for novel nucleic acid binding proteins present in cell extracts of T. tenax. An assay for proteins capable of binding to a single-stranded DNA oligonucleotide resulted in identification of three proteins. The first protein, Alba, has been shown previously to bind single-stranded DNA as well as duplex DNA. The two other proteins, which we designated CC1 (for crenarchaeal chromatin protein 1), are very closely related to one another, and homologs are restricted to the P. aerophilum and Aeropyrum pernix genomes. CC1 is a 6-kDa, monomeric, basic protein that is expressed at a high level in T. tenax. This protein binds single- and double-stranded DNAs with similar affinities. These properties are consistent with a role for CC1 as a crenarchaeal chromatin protein. 相似文献
5.
The purification to homogeneity of the non-heme iron protein, sometimes referred to as either "red protein" or "paramagnetic protein", from Clostridium pasteurianum W5 extracts is described and its physicochemical properties studied. This paramagnetic protein (g= 1.94) has a molecular weight of about 25000 and contains two iron and two acid-labile sulfur atoms per mol of protein. Its midpoint potential at pH 7.5, as determined by electron paramagnetic resonance titration, is -300 mV. Optical circular dichroism and electron paramagnetic resonance spectra of the paramagnetic protein are similar to those of two iron-two acid-labile sulfur ferredoxins. The biochemical reduction of the purified protein was also studied. 相似文献
6.
《Journal of molecular graphics》1990,8(1):11-15
MATRIX is a program designed primarily to enable the user to visualize all regions of similarity between two proteins at a glance. The program helps the user to see where they are similar—at what relative positions in the amino acid sequences of the two proteins in question does the similarity exist; how they are similar—what functional characteristics the two similar sequences have in common; and to what extent they are similar—is the similarity significant, if so how significant relative to other similar sequences in the protein. This is achieved by constructing a diagram in which quantitative parameters of amino acids are used to compare every amino acid residue of the first protein with every amino acid residue of the second.Another function of the program is, given two sets of atomic coordinates—either of different proteins or for the same protein (for self-comparison)—to demonstrate which residues of the two proteins, when the two proteins are superimposed upon each other, appear in the same space (or are close to each other). 相似文献
7.
Biochemical basis of the temperature-inducible constitutive protease activity of the RecA441 protein of Escherichia coli 总被引:1,自引:0,他引:1
We compared the biochemical properties of the RecA441 protein to those of the wild-type RecA protein in an effort to account for the constitutive protease activity observed in recA441 strains. The two RecA proteins have similar properties in the absence of single-stranded DNA binding protein (SSB protein), and the differences that do exist shed little light on the temperature-inducible phenotype observed in recA441 strains. In contrast, several biochemical differences are apparent when the two proteins are compared in the presence of SSB protein, and these are conducive to a hypothesis that explains the temperature-sensitive behavior observed in these strains. We find that both the single-stranded DNA (ssDNA)-dependent ATPase and LexA-protease activities of RecA441 protein are more resistant to inhibition by SSB protein than are the activities of the wild-type protein. Additionally, the RecA441 protein is more capable of using ssDNA that has been precoated with SSB protein as a substrate for ATPase and protease activities, implying that RecA441 protein is more proficient at displacing SSB protein from ssDNA. The enhanced SSB protein displacement ability of the RecA441 protein is dependent on elevated temperature. These observations are consistent with the hypothesis that the RecA441 protein competes more efficiently with SSB protein for limited ssDNA sites and can be activated to cleave repressors at elevated temperature by displacing SSB protein from the limited ssDNA that occurs naturally in Escherichia coli. Neither the ssDNA binding characteristics of the RecA441 protein nor the rate at which it transfers from one DNA molecule to another provides an explanation for its enhanced activities, leading us to conclude that kinetics of RecA441 protein association with DNA may be responsible for the properties of the RecA441 protein. 相似文献
8.
Identification and characterization of cellular targets for tyrosine protein kinases 总被引:20,自引:0,他引:20
Protein kinases associated with the transforming proteins of a number of retroviruses are specific for tyrosine. Several proteins in cells transformed by these viruses are phosphorylated at tyrosine. We have now identified three unrelated abundant nonphosphorylated cellular proteins of 46,000, 39,000 and 28,000 daltons in chick embryo cells, which are the unphosphorylated forms of phosphotyrosine-containing proteins and thus are substrates for tyrosine protein kinases. By two-dimensional gel analysis, we have found that the 46,000-dalton protein exists in two unphosphorylated forms of which the more acidic is a minor species. This latter form is phosphorylated, chiefly at serine, in both normal and transformed cells, generating a yet more acidic species. In transformed but not normal cells, the major form is phosphorylated at tyrosine and serine, yielding a fourth isoelectric variant. The 46,000-dalton unphosphorylated protein has been partially purified, and antiserum to it recognizes all four isoelectric variants of the protein. The 39,000-dalton protein has two unphosphorylated forms of which the more acidic is a minor species. The major form is phosphorylated at tyrosine and serine in transformed cells only. The 39,000-dalton unphosphorylated protein has been partially purified, and antiserum raised to it recognizes all three isoelectric variants. The 28,000-dalton protein has a single phosphorylated form which contains serine in normal cells, but both serine and tyrosine in transformed cells. 相似文献
9.
Chick brain microtubule protein consists primarily of a mixture of MAP2:tubulin oligomers and dimeric tubulin. The assembly of this protein is described by a single pseudofirst-order reaction at 20 microM GTP, but by the summation of two pseudofirst-order reactions at 1 mM GTP. The protein contains two GTP-binding species, corresponding to the tubulin dimers and the oligomers, and conditions which alter the dimer: oligomer equilibrium, affect the kinetics of microtubule assembly. The results indicate that the oligomers are only direct assembly intermediates at high GTP concentrations. 相似文献
10.
Structure and expression of a Xenopus gene encoding an snRNP protein (U1 70K). 总被引:22,自引:3,他引:19
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M Etzerodt R Vignali G Ciliberto D Scherly I W Mattaj L Philipson 《The EMBO journal》1988,7(13):4311-4321
11.
Dynamics and unfolding pathways of a hyperthermophilic and a mesophilic rubredoxin. 总被引:6,自引:2,他引:4
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T. Lazaridis I. Lee M. Karplus 《Protein science : a publication of the Protein Society》1997,6(12):2589-2605
Molecular dynamics simulations in solution are performed for a rubredoxin from the hyperthermophilic archaeon Pyrococcus furiosus (RdPf) and one from the mesophilic organism Desulfovibrio vulgaris (RdDv). The two proteins are simulated at four temperatures: 300 K, 373 K, 473 K (two sets), and 500 K; the various simulations extended from 200 ps to 1,020 ps. At room temperature, the two proteins are stable, remain close to the crystal structure, and exhibit similar dynamic behavior; the RMS residue fluctuations are slightly smaller in the hyperthermophilic protein. An analysis of the average energy contributions in the two proteins is made; the results suggest that the intraprotein energy stabilizes RdPf relative to RdDv. At 373 K, the mesophilic protein unfolds rapidly (it begins to unfold at 300 ps), whereas the hyperthermophilic does not unfold over the simulation of 600 ps. This is in accord with the expected stability of the two proteins. At 473 K, where both proteins are expected to be unstable, unfolding behavior is observed within 200 ps and the mesophilic protein unfolds faster than the hyperthermophilic one. At 500 K, both proteins unfold; the hyperthermophilic protein does so faster than the mesophilic protein. The unfolding behavior for the two proteins is found to be very similar. Although the exact order of events differs from one trajectory to another, both proteins unfold first by opening of the loop region to expose the hydrophobic core. This is followed by unzipping of the beta-sheet. The results obtained in the simulation are discussed in terms of the factors involved in flexibility and thermostability. 相似文献
12.
Rat lymph chylomicrons were separated into two fractions using heparin-Sepharose chromatography: a major fraction which elutes from the column with the void volume at 0.05 M NaCl, and a smaller fraction which binds to the column at 0.05 M NaCl and elutes at 0.3 M NaCl. These two fractions differ in mean particle size, and lipid and protein compositions. Both fractions share apolipoproteins B, A-IV, E, A-I, and C, but the fraction which binds to heparin-Sepharose contains two additional proteins: protein I (Mr = 6.0 X 10(4)), and protein II (Mr = 8.0 X 10(4)). Both proteins are also present in the lipoprotein-free fraction of rat serum. Proteins I and II bind to heparin-Sepharose, and are highly amphiphilic: they bind with high affinity to phospholipid surfaces and form stable monolayers at the air-water interface. The molecular weight, amino acid composition, heparin binding, and amphiphilicity of protein I resemble that of beta 2-glycoprotein I; in addition, protein I from rat lymph chylomicrons cross-reacts with rabbit antiserum to human beta 2-glycoprotein I, suggesting that these two proteins are homologous. Protein II appears to be a previously undescribed protein. The possible functions of these two proteins are discussed. 相似文献
13.
The ultraviolet visible, and near infrared spectrum of a two-iron protein from Desulphovibrio gigas, a new type of non-haem iron protein lacking labile sulphide, is compared with that of D. gigas rubredoxin. The charge transfer band maxima of rubredoxin at 495 and 565 nm are less separated in the new protein implying a higher symmetry of the two iron centres. The existence of a spin-spin interaction between the two iron centres in the new protein is suggested by the magnetic susceptibility measurements of the oxidized and reduced states of both proteins, which gives a smaller value per iron centre for the new protein. The oxidized form of the two iron-protein has a complex EPR spectrum with signals at g values of 8.97, 7.72, 5.73, 4.94, and 1.84. An EPR titration gives a value of --35 +/- 15 mV for the two signals at g values of 7.72 and 5.73. Rubredoxin has the characteristic spectrum of rubredoxins with two signals at g values of 9.4 and 4.27. 相似文献
14.
Abstract— The molecular weight of β nerve growth factor protein determined by sedimentation equilibrium in sodium acetate buffer, pH 40, and at protein concentrations around 0-5 mg/ml agrees with the value obtained from the amino acid sequence and confirms the dimeric character of the protein under these conditions. At pH values of 5.0 or greater, β nerve growth factor protein shows either partial dissociation into monomers or aggregation to higher polymers or both phenomena. The extent of dissociation or aggregation depends on buffer type and pH and is most pronounced at alkaline pH. The variation of molecular weight of β nerve growth factor with solvent conditions is similar to that of insulin or proinsulin. Removal of either the two COOH-terminal arginine residues or the two NH2 -terminal octapeptide sequences from the protein has no effect on its solution properties at acid pH, the protein remaining a dimer. Species such as 2-5 S nerve growth factor or cyanogen bromide cleaved nerve growth factor which are partically deficient in COOH-terminal arginine residues and/or NH2 -octapeptide or nonapeptide sequences are also dimers at pH40. The protein derivative which lacks the two NH2 -terminal octapeptide sequence does not, like β-nerve growth factor, display dissociation or aggregation behavior at neutral pH, indicating that these sequences are involved in monomer-monomer interactions. 相似文献
15.
The two Ni2+ ions in the urease active site are delivered by the metallochaperone UreE, whose metal binding properties are central to the assembly of this metallocenter. Isothermal titration calorimetry (ITC) has been used to quantify the stoichiometry, affinity, and thermodynamics of Ni2+, Cu2+, and Zn2+ binding to the well-studied C-terminal truncated H144*UreE from Klebsiella aerogenes, Ni2+ binding to the wild-type K. aerogenes UreE protein, and Ni2+ and Zn2+ binding to the wild-type UreE protein from Bacillus pasteurii. The stoichiometries and affinities obtained by ITC are in good agreement with previous equilibrium dialysis results, after differences in pH and buffer competition are considered, but the concentration of H144*UreE was found to have a significant effect on metal binding stoichiometry. While two metal ions bind to the H144*UreE dimer at concentrations <10 microM, three Ni2+ or Cu2+ ions bind to 25 microM dimeric protein with ITC data indicating sequential formation of Ni/Cu(H144*UreE)4 and then (Ni/Cu)2(H144*UreE)4, or Ni/Cu(H144*UreE)2, followed by the binding of four additional metal ions per tetramer, or two per dimer. The thermodynamics indicate that the latter two metal ions bind at sites corresponding to the two binding sites observed at lower protein concentrations. Ni2+ binding to UreE from K. aerogenes is an enthalpically favored process but an entropically driven process for the B. pasteurii protein, indicating chemically different Ni2+ coordination to the two proteins. A relatively small negative value of DeltaCp is associated with Ni2+ and Cu2+ binding to H144*UreE at low protein concentrations, consistent with binding to surface sites and small changes in the protein structure. 相似文献
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The three-dimensional structure of the hybrid cluster protein from Desulfovibrio vulgaris (Hildenborough) has been determined at 1.6 A resolution using synchrotron X-ray radiation. The protein can be divided into three domains: an N-terminal mainly alpha-helical domain and two similar domains comprising a central beta-sheet flanked by alpha-helices. The protein contains two 4Fe clusters with an edge-to-edge distance of 10.9 A. Four cysteine residues at the N-terminus of the protein are ligands to the iron atoms of a conventional [4Fe-4S] cubane cluster. The second cluster has an unusual asymmetric structure and has been named the hybrid cluster to reflect the variety of protein ligands, namely two mu-sulfido bridges, two mu(2)-oxo bridges, and a further disordered bridging ligand. Anomalous differences in data collected at 1.488 A and close to the iron edge at 1.743 A have been used to confirm the identity of the metal and sulfur atoms. The hybrid cluster is buried in the center of the protein, but is accessible through a large hydrophobic cavity that runs the length of domain 3. Hydrophobic channels have previously been identified as access routes to the active centers in redox enzymes with gaseous substrates. The hybrid cluster is also accessible by a hydrophilic channel. The [4Fe-4S] cubane cluster is close to an indentation on the surface of the protein and can also be approached on the opposite side by a long solvent channel. At the present time, neither the significance of these channels nor, indeed, the function of the hybrid cluster protein is known. 相似文献
18.
Molecular basis for the differential subcellular localization of the 38- and 39-kilodalton structural proteins of Borna disease virus. 总被引:3,自引:2,他引:1
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Borna disease virus (BDV) is a nonsegmented negative-strand (NNS) RNA virus that is unusual because it replicates in the nucleus. The most abundant viral protein in infected cells is a 38/39-kDa doublet that is presumed to represent the nucleocapsid. Infectious particles also contain high levels of this protein, accounting for at least 50% of the viral proteins. The two forms of the protein differ by an additional 13 amino acids that are present at the amino terminus of the 39-kDa form and missing from the 38-kDa form. To examine whether this difference in amino acid content affects the localization of this protein in cells, the 39- and 38-kDa proteins were expressed in transfected cells. The 39-kDa form was concentrated in the nucleus, whereas the 38-kDa form was found in both the nucleus and cytoplasm. Inspection of the extra 13 amino acids present in the 39-kDa form revealed a sequence (Pro-Lys-Arg-Arg) that is very similar to the nuclear localization signals (in both sequence homology and amino-terminal location) of the VP1 proteins of simian virus 40 and polyomavirus. Primer extension analysis of total RNA from infected cells suggests that there are two mRNA species encoding the two forms of the nucleocapsid protein. In infected cells, the 39-kDa form is expressed at about twofold-higher levels than the 38-kDa form at both the RNA and protein levels. The novel nuclear localization of the 39-kDa nucleocapsid-like protein suggests that this form of the protein is targeted to the nucleus, the site for viral RNA replication, and that it may associate with genomic RNA. 相似文献
19.
Cell-cycle-specific inhibition by chloramphenicol of septum fromation and cell division in synchronized cells of Bacillus subtilis. 总被引:6,自引:5,他引:1
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The relationship between protein synthesis and processes of cell division was studied by using synchronized cells of Bacillus subtilis 168. The addition of chloramphenicol at the beginning of synchronous growth prevented septum formation and cell division, suggesting the requirement of protein synthesis for the processes of cell division. Experiments in which the drug was added to the cells at different cell ages showed that the protein synthesis required for the initiation of septum formation was completed at about 15 min and that the protein synthesis required for cell division was completed at about 45 min. By interpreting the result from the concept of the transition point for protein synthesis, it was suggested that the processes of cell division in B. subtilis require at least two kinds of protein molecules which are synthesized at distinct stages in the cell cycle. This was supported by the result of an experiment in which starvation and the readdition of a required amino acid to exponentially growing cells induced two steps of synchronous cell division. Further, the two transition points are in agreement with the estimations obtained by residual division after the inhibition of protein synthesis in asynchronous cells. The relationship of the timing between the completion of chromosome replication and the two transition points was also studied. 相似文献
20.
A double mutant cycle (DMC) approach was employed to estimate the effect of temperature on the contribution of two highly conserved salt bridges to protein stability in the hyperthermophilic protein Ssh10b. The coupling free energy were 2.4 +/- 0.4 kJ/mol at 298 K and 2.2 +/- 0.4 kJ/mol at 353 K for Glu-54/Arg-57, and 6.0 +/- 0.2 kJ/mol at 298 K and 5.9 +/- 0.6 kJ/mol at 353 K for Glu-36/Lys-68. The stability free energy of Ssh10b decrease greatly with increasing temperature, while the direct contribution of these two salt bridges to protein stability remain almost constant, providing evidence supporting the theoretical prediction that salt bridges are extremely resilient to temperature increases and thus are specially suited to improving protein stability at high temperatures. The reason for the difference in coupling free energy between salt bridges Glu-54/Arg-57 and Glu-36/Lys-68 is discussed. Comparing our results with published DMC data for the contribution of salt bridges to stability in other proteins, we found that the energy contribution of a salt bridge formed by two charged residues far apart in the primary sequence is higher than that of those formed between two very close ones. Implications of this finding are useful for engineering proteins with enhanced thermostability. 相似文献