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1.
Deletion mutants of arginine kinase (AK) were constructed: AKND4, AKND6, AKND8, AKND10 (the first 4, 6, 8 and 10 amino acids of the N-terminal were deleted), to investigate the structural and functional roles of the N-terminal. Results showed that the deletion mutants assume less compact conformations compared to the wild-type, whereas no significant changes of the secondary or the quaternary structures were observed, implying that the deletions cause a perturbation in the tertiary structure or the hydrodynamic properties of the enzyme. The enzymatic and denaturing measurements showed that removal of the N-terminal residues decreased the activity and stability of the enzyme markedly. The instability increased in accord with the increased number of amino acid residues removed from the N-terminal of AK. It can be concluded that the N-terminal of AK plays an important role in maintaining the conformational stability and catalytic function of the enzyme.  相似文献   

2.
探讨FⅩⅢ A基因表达的分子机制.凝胶阻滞实验(EMSA)结果证明,FⅩⅢ A基因第1内含子5′区的前12碱基与转录因子结合并由此调控基因表达.FⅩⅢ A基因第1内含子第12位碱基由C突变为A(内含子1 (+12)C→A)导致与转录因子结合能力降低.构建不同的荧光素酶表达质粒Luc 1、Luc 2、Luc 3、Luc 4、Luc 5、Luc 6,并转染U937细胞和HepG2细胞.结果显示,如果Luc 5(具有最高表达活性)的内含子1(+12)由C突变为A,启动子活性显著降低.与Luc 5相比,突变后的Luc 5的活性分别下降了52.9%(U937, P<0.01)和47.6%(HepG2, P<0.01).将Luc 5与PN3 Sp1共同转染U937细胞和HepG2细胞后,Luc 5的荧光素酶活性分别提高了42.4%(U937,与Luc 5单独转染比较P<0.01)和54.9%(HepG2, 与Luc 5单独转染比较P<0.01),而突变后的Luc 5(Mut)与PN3 Sp1共同转染则没有明显的改变.表明转录因子Sp1在FⅩⅢ A基因表达的重要性.这些结果也表明,内含子在FⅩⅢ A基因表达过程中起重要作用,为遗传性凝血因子发病机理的研究提供了新的证据.  相似文献   

3.
We have constructed and analyzed amino terminally deleted analogs of IL-6. Progressively shortened variants of mature IL-6 were constructed at the cDNA level and expressed in Escherichia coli. Mutant proteins were recovered from refractile bodies by solubilizing in 6 M guanidine-HCl. The mutant protein concentration in these preparations was estimated by Western blotting by using an IL-6-specific mAb and the biologic activity was measured in the B9 (hybridoma growth factor) assay. The first 28 amino acids of mature IL-6 could be removed without significantly affecting biologic activity. A further removal of amino acids 29 and 30 resulted in an approximately 50-fold decrease, whereas removal of amino acids 31 to 34 virtually abolished the activity. The mutants showed the same reaction pattern in three other IL-6 assays: induction of murine thymocyte proliferation, induction of fibrinogen synthesis by a human hepatoma cell line (HepG2), and the induction of IgM synthesis by an EBV-transformed B cell line. This suggests that a single functional domain might be responsible for all four activities of IL-6.  相似文献   

4.
Li L  Liu L  Hong R  Robertson D  Hastings JW 《Biochemistry》2001,40(6):1844-1849
Gonyaulax luciferase is a single-chain ( approximately 137 kDa) polypeptide comprising 111 N-terminal amino acids followed by three contiguous homologous domains (377 amino acids each). Each domain has luciferase activity, accounting for the earlier observation that proteolytic fragments ( approximately 35 kDa) of luciferase are active. The activity of the full-length native enzyme is maximal at pH 6.3, dropping to near zero at pH 8; the activity of fragments also peaks at pH 6.3 but remains high at 8. While the activity loss at higher pH might be thought to be associated with the conformation of the full-length protein, we show here that this is a property of individual domains. The three intramolecularly homologous domains, separately cloned and expressed in Escherichia coli as fusion proteins, exhibit pH-activity curves similar to that of the full-length enzyme. For each domain the removal of approximately 50 N-terminal amino acids resulted in an increase in the ratio of luciferase activity at pH 8 relative to that at pH 6.3, such that their pH-activity profiles mimicked that of the proteolytic fragments reported earlier. Replacement of N-terminal histidines by alanine by site-directed mutagenesis identified four that are involved in the loss of activity at high pH. This system illustrates an unusual, possibly unique mechanism for pH regulation of enzyme activity, which has been postulated to be responsible for the control of the characteristic flashes of bioluminescence.  相似文献   

5.
The molecular chaperone Hsp90 is required for the folding and activation of a large number of substrate proteins. These are involved in essential cellular processes ranging from signal transduction to viral replication. For the activation of its substrates, Hsp90 binds and hydrolyzes ATP, which is the key driving force for conformational conversions within the dimeric chaperone. Dimerization of Hsp90 is mediated by a C-terminal dimerization site. In addition, there is a transient ATP-induced dimerization of the two N-terminal ATP-binding domains. The resulting ring-like structure is thought to be the ATPase-active conformation. Hsp90 is a slow ATPase with a turnover number of 1 ATP/min for the yeast protein. A key question for understanding the molecular mechanism of Hsp90 is how ATP hydrolysis is regulated and linked to conformational changes. In this study, we analyzed the activation process structurally and biochemically with a view to identify the conformational limitations of the ATPase reaction cycle. We showed that the first 24 amino acids stabilize the N-terminal domain in a rigid state. Their removal confers flexibility specifically to the region between amino acids 98 and 120. Most surprisingly, the deletion of this structure results in the complete loss of ATPase activity and in increased N-terminal dimerization. Complementation assays using heterodimeric Hsp90 show that this rigid lid acts as an intrinsic kinetic inhibitor of the Hsp90 ATPase cycle preventing N-terminal dimerization in the ground state. On the other hand, this structure acts, in concert with the 24 N-terminal amino acids of the other N-terminal domain, to form an activated ATPase and thus regulates the turnover number of Hsp90.  相似文献   

6.
To investigate the functional domains involved in the biological activity of staphylococcal enterotoxin (SEC2), a series of SEC2 mutants were constructed. Deletion of the last 77 amino acids at the C-terminus of SEC2 did not affect its native superantigen and fever activities, and further removal of the C-terminal residues reduced SEC2 activities significantly. On the other hand, the mutants lacking 18 or more N-terminal residues severely impaired superantigen activity. These data indicated that the functional regions for the biological activities of SEC2 were confined to N-terminal domain, further implied that the proper three-dimensional structure of SEC2 is not needed for its biological activities. Our results deliver valuable information that it is possible to design new SEC2 immunotherapeutic agents which have the superantigen activity and low molecular weight for permeability.  相似文献   

7.
In this report, we describe plasmids that direct the expression of active mouse interleukin 2 (mIL 2) in Escherichia coli, and the use of this expression system to perform a mutational analysis of the N-terminal region of the mIL 2 protein. We found that the N-terminus was tolerant to the addition of a few amino acids, and even the addition of 20 amino acids resulted in only a modest decrease in activity of the protein. The bioactivity of mIL 2 as defined by its ability to sustain the proliferation of cloned T cells was also only minimally affected by deletion of up to 13 N-terminal amino acids, or of the entire poly-GLN stretch (amino acids 15-26). Deletion of the 30 N-terminal amino acids drastically reduced but did not abolish activity. Deletion of the 41 N-terminal amino acids completely abolished activity, whereas certain changes in the initial 37 amino acids drastically reduced the biological activity of the protein. We also analyzed the immunoreactivity of the mutant proteins with the anti-IL 2 monoclonal antibodies S4B6 and DMS-1. This analysis showed that the determinant recognized by S4B6 required that the N-terminal mIL 2 amino acids 26-45 be intact, whereas the DMS-1 determinant was located to the C-terminal side of amino acid 46.  相似文献   

8.
Zhang Z  Rowe J  Wang W  Sommer M  Arvin A  Moffat J  Zhu H 《Journal of virology》2007,81(17):9024-9033
To efficiently generate varicella-zoster virus (VZV) mutants, we inserted a bacterial artificial chromosome (BAC) vector in the pOka genome. We showed that the recombinant VZV (VZV(BAC)) strain was produced efficiently from the BAC DNA and behaved indistinguishably from wild-type virus. Moreover, VZV's cell-associated nature makes characterizing VZV mutant growth kinetics difficult, especially when attempts are made to monitor viral replication in vivo. To overcome this problem, we then created a VZV strain carrying the luciferase gene (VZV(Luc)). This virus grew like the wild-type virus, and the resulting luciferase activity could be quantified both in vitro and in vivo. Using PCR-based mutagenesis, open reading frames (ORF) 0 to 4 were individually deleted from VZV(Luc) genomes. The deletion mutant viruses appeared after transfection into MeWo cells, except for ORF4, which was essential. Growth curve analysis using MeWo cells and SCID-hu mice indicated that ORF1, ORF2, and ORF3 were dispensable for VZV replication both in vitro and in vivo. Interestingly, the ORF0 deletion virus showed severely retarded growth both in vitro and in vivo. The growth defects of the ORF0 and ORF4 mutants could be fully rescued by introducing wild-type copies of these genes back into their native genome loci. This work has validated and justified the use of the novel luciferase VZV BAC system to efficiently generate recombinant VZV variants and ease subsequent viral growth kinetic analysis both in vitro and in vivo.  相似文献   

9.
Reconstitution experiments with a chemically synthesized core light-harvesting (LH1) beta-polypeptide analogue having 3-methylhistidine instead of histidine in the position that normally donates the coordinating ligand to bacteriochlorophyll (Bchl) have provided the experimental data needed to assign to B820 one of the two possible alphabeta.2Bchl pairs that are observed in the crystal structure of LH2 from Phaeospirillum (formerly Rhodospirillum) molischianum, the one with rings III and V of Bchl overlapping. Consistent with the assigned structure, experimental evidence is provided to show that significant stabilizing interactions for both the subunit complex (B820) and LH1 occur between the N-terminal regions of the alpha- and beta-polypeptides. On the basis of the results with the chemically synthesized polypeptides used in this study, along with earlier results with protease-modified polypeptides, mutants, and chemically synthesized polypeptides, the importance of a stretch of 9-13 amino acids at the N-terminal end of the alpha- and beta-polypeptides is underscored. A progressive loss of interaction with the LH1 beta-polypeptide was found as the first three N-terminal amino acids of the LH1 alpha-polypeptide were removed. The absence of the N-terminal formylmethionine (fMet), or conversion of the sulfur in this fMet to the sulfoxide, resulted in a decrease in LH1 formation. In addition to the removal of fMet, removal of the next two amino acids also resulted in a decrease in K(assoc) for B820 formation and nearly eliminated the ability to form LH1. It is suggested that the first three amino acids (fMetTrpArg) of the LH1 alpha-polypeptide of Rhodospirillum rubrum form a cluster that is most likely involved in close interaction with the side chain of His -18 (see Figure 1 for numbering of amino acids) of the beta-polypeptide. The results provide evidence that the folding motif of the alpha- and beta-polypeptides in the N-terminal region observed in crystal structures of LH2 is also present in LH1 and contributes significantly to stabilizing the complex.  相似文献   

10.
Deletion mutants of rabbit muscle lactate dehydrogenase (LDH) were constructed using polymerase chain reaction (PCR) to study the roles of N-terminal residues. The coding sequences of the first 5 (LD5) and 10 (LD10) amino acids of the N-terminus were deleted and the gene was inserted into the prokaryotic expression vector pET21b. The mutant enzymes were expressed in E. coli BL21/DE3 and were purified. Then their characteristics and stabilities were studied. The results showed LDH was completely inactivated when the first 10 N-terminal amino acid residues were removed, but the mutant (LD10) could have partially restored activity in the presence of structure-making ions. The removal of the first 5 and 10 N-terminal amino acid residues did not affect the aggregation state of the enzyme, that is, LD5 and LD10 were still tetramers. The stabilities of recombinant wild-type LDH (RW-LD), LD5, and LD10 were compared by incubating them at low pH, elevated temperature, and high GuHCl. The results showed that the N-terminal deletion mutants were more sensitive to denaturing environments; they were easily inactivated and unfolded. Their instability increased and their ability to refold decreased with the increased number of amino acid residues removed from the N-terminus of LDH. These results confirm that the N-terminus of LDH plays a crucial role in stabilizing the structure and in maintaining the function of the enzyme.  相似文献   

11.
Mutagenesis of human granulocyte colony stimulating factor   总被引:9,自引:0,他引:9  
To define the structure-function relationship, we have made a number of mutants of human granulocyte colony-stimulating factor (hG-CSF) by in vitro mutagenesis. The results indicate that most of the mutations located in the internal and C-terminal regions of the molecule abolished the activity, whereas the mutants without N-terminal 4, 5, 7, or 11 amino acids retained the activity. N-terminal amino acids were also altered by cassette mutagenesis using a synthetic oligonucleotide mixture. Among them, KW2228, in which Thr-1, Leu-3, Gly-4, Pro-5 and Cys-17 were respectively substituted with Ala, Thr, Tyr, Arg and Ser, showed more potent granulopoietic activity than that of intact hG-CSF both in vitro and in vivo.  相似文献   

12.
Tanaka N  Tani Y  Tada T  Lee YF  Kanaori K  Kunugi S 《Biochemistry》2006,45(28):8556-8561
Escherichia coli heat shock protein ClpB disaggregates denatured protein in cooperation with the DnaK chaperone system. Several studies showed that the N-terminal domain is essential for the chaperone activity, but its role is still largely unknown. The N-terminal domain contains two structurally similar subdomains, and conserved amino acids Thr7 and Ser84 share the same position in two apparent sequence repeats. T7A and S84A substitutions affected chaperone activity of ClpB without significantly changing the native conformation [Liu, Z. et al. (2002) J. Mol. Biol. 321, 111-120]. In this study, we aimed to better understand the roles of several conserved amino acid residues, including Thr7 and Ser84, in the N-terminal domain. We investigated the effects of mutagenesis on substrate binding and conformational states of ClpB N-terminal domain fragment (ClpBN). Fluorescence polarization analysis showed that the T7A and S84A substitutions enhanced the interaction between ClpBN and protein aggregates. Interestingly, further analyses suggested that the mechanisms by which they do so are quite different. For T7A substitution, the increased substrate affinity could be due to a conformational change in the hydrophobic core as revealed by NMR spectroscopy. In contrast, for S84A, increased substrate binding would be explained by a unique conformational state of this mutant as revealed by pressure perturbation analysis. The thermal transition temperature of the S84A mutant, monitored by DSC, was 6.1 degrees C lower than that of wild-type. Our results revealed that conserved amino acids Thr7 and Ser84 both participated in maintaining the conformational integrity of the ClpB N-terminal domain.  相似文献   

13.
UDP-GlcNAc : -3-D-mannoside -1,2-N-acetylglucosaminyltransferase I (GnT I, EC 2.4.1.101) plays an essential role in the conversion of oligomannose to complex and hybrid N-glycans. Rabbit GnTI is 447 residues long and has a short four-residue N-terminal cytoplasmic tail, a 25-residue putative signal–anchor hydrophobic domain, a stem region of undetermined length and a large C-terminal catalytic domain, a structure typical of all glycosyltransferases cloned to date. Comparison of the amino acid sequences for human, rabbit, mouse, rat, chicken, frog and Caenorhabditis elegans GnT I was used to obtain a secondary structure prediction for the enzyme which suggested that the location of the junction between the stem and the catalytic domain was at about residue 106. To test this hypothesis, several hybrid constructs containing GnT I with N- and C-terminal truncations fused to a mellitin signal sequence were inserted into the genome of Autographa californica nuclear polyhedrosis virus (AcMNPV), Sf 9 insect cells were infected with the recombinant baculovirus and supernatants were assayed for GnT I activity. Removal of 29, 84 and 106 N-terminal amino acids had no effect on GnT I activity; however, removal of a further 14 amino acids resulted in complete loss of activity. Western blot analysis showed strong protein bands for all truncated enzymes except for the construct lacking 120 N-terminal residues indicating proteolysis or defective expression or secretion of this protein. The data indicate that the stem is at least 77 residues long.  相似文献   

14.
Firefly luciferase is a monomeric protein composed of two globular domains. There is a wide cleft between the two domains. The N-terminal domain can be further divided into A-, B-, and C-subdomains. Previous studies showed that in vitro unfolding of firefly luciferase induced by guanidinium chloride can be described as a four-state equilibrium with two inactive intermediates (Herbst, R., et al. (1997) J. Biol. Chem. 272, 7099-7105). In order to monitor spectroscopically the conformational changes that occur in the different domains and subdomains during the multi-state unfolding process, we constructed a series of single-tryptophan mutants. These mutants were purified and characterized and shown to retain essentially all of the structural properties of the wild-type luciferase. Under equilibrium conditions, the unfolding of each mutant protein were studied by means of fluorescence and circular dichroism. The results show that different conformational changes occur in specific regions, suggesting a sequential unfolding process for firefly luciferase. Under 2.5 M GdmCl, whereas the N-domain unfolds partially holding half of the secondary structure content, the C-domain unfolds almost completely. In the equilibrium intermediate I(2), the secondary structure might stem mostly from the A- and B- subdomains.  相似文献   

15.
Heme Nitric oxide/OXygen binding (H-NOX) domains have provided a novel scaffold to probe ligand affinity in hemoproteins. Mutation of isoleucine 5, a conserved residue located in the heme-binding pocket of the H-NOX domain from Thermoanaerobacter tengcongensis (Tt H-NOX), was carried out to examine changes in oxygen (O(2))-binding properties. A series of I5 mutants (I5F, I5F/I75F, I5F/L144F, I5F/I75F/L144F) were investigated to probe the role of steric bulk within the heme pocket. The mutations significantly increased O(2) association rates (1.5-2.5-fold) and dissociation rates (8-190-fold) as compared to wild-type Tt H-NOX. Structural changes that accompanied the I5F mutation were characterized using X-ray crystallography and resonance Raman spectroscopy. A 1.67 ? crystal structure of the I5F mutant indicated that introducing a phenylalanine at position 5 resulted in a significant shift of the N-terminal domain of the protein, causing an opening of the heme pocket. This movement also resulted in an increased amount of flexibility at the N-terminus and the loop covering the N-terminal helix as indicated by the two conformations of the first six N-terminal amino acids, high B-factors in this region of the protein, and partially discontinuous electron density. In addition, introduction of a phenylalanine at position 5 resulted in increased flexibility of the heme within the pocket and weakened hydrogen bonding to the bound O(2) as measured by resonance Raman spectroscopy. This study provides insight into the critical role of I5 in controlling conformational flexibility and ligand affinity in H-NOX proteins.  相似文献   

16.
The substituted cysteine accessibility approach, combined with chemical modification using membrane-impermeant alkylating reagents, was used to identify functionally important structural elements of the rat type IIa Na(+)/P(i) cotransporter protein. Single point mutants with different amino acids replaced by cysteines were made and the constructs expressed in Xenopus oocytes were tested for function by electrophysiology. Of the 15 mutants with substituted cysteines located at or near predicted membrane-spanning domains and associated linker regions, 6 displayed measurable transport function comparable to wild-type (WT) protein. Transport function of oocytes expressing WT protein was unchanged after exposure to the alkylating reagent 2-aminoethyl methanethiosulfonate hydrobromide (MTSEA, 100 microM), which indicated that native cysteines were inaccessible. However, for one of the mutants (S460C) that showed kinetic properties comparable with the WT, alkylation led to a complete suppression of P(i) transport. Alkylation in 100 mM Na(+) by either cationic ([2-(trimethylammonium)ethyl] methanethiosulfonate bromide (MTSET), MTSEA) or anionic [sodium(2-sulfonatoethyl)methanethiosulfonate (MTSES)] reagents suppressed the P(i) response equally well, whereas exposure to methanethiosulfonate (MTS) reagents in 0 mM Na(+) resulted in protection from the MTS effect at depolarized potentials. This indicated that accessibility to site 460 was dependent on the conformational state of the empty carrier. The slippage current remained after alkylation. Moreover, after alkylation, phosphonoformic acid and saturating P(i) suppressed the slippage current equally, which indicated that P(i) binding could occur without cotransport. Pre-steady state relaxations were partially suppressed and their kinetics were significantly faster after alkylation; nevertheless, the remaining charge movement was Na(+) dependent, consistent with an intact slippage pathway. Based on an alternating access model for type IIa Na(+)/P(i) cotransport, these results suggest that site 460 is located in a region involved in conformational changes of the empty carrier.  相似文献   

17.
The structural gene of streptolysin O was cloned from Streptococcus pyogenes strain Sa and S. equisimilis H46A, and the nucleotide sequences were compared with those of strain Richards. To obtain the minimal active fragment of the toxin and to elucidate structure-function relationships in hemolytic function, streptolysin O mutants deleted in N- and C-terminal regions were constructed. Internal amino acid residues were also replaced by introduction of point mutations. Analyses of these mutants showed that considerable activity was retained even after deletion of the N-terminal 107 residues, but genetic removal of the ultimate C-terminal residue resulted in a marked decrease in hemolytic function. By removal in succession, hemolytic activity declined exponentially, and only 0.002% of the activity remained after deletion of the C-terminal four residues. Nucleotide replacement experiments indicated pivotal roles of I202, V217, D324-L325, V339, and H469 residues in hemolysis.  相似文献   

18.
19.
20.
Restrictocin, produced by the fungus Aspergillus restrictus, is a highly specific ribonucleolytic toxin which cleaves a single phosphodiester bond between G4325 and A4326 in the 28S rRNA. It is a nonglycosylated, single-chain, basic protein of 149 amino acids. The putative catalytic site of restrictocin includes Tyr47, His49, Glu95, Arg120 and His136. To map the catalytic activity in the restrictocin molecule, and to study the role of N- and C-terminus in its activity, we have systematically deleted amino-acid residues from both the termini. Three N-terminal deletions removing 8, 15 and 30 amino acids, and three C-terminal deletions lacking 4, 6, and 11 amino acids were constructed. The deletion mutants were expressed in Escherichia coli, purified to homogeneity and functionally characterized. Removal of eight N-terminal or four C-terminal amino acids rendered restrictocin partially inactive, whereas any further deletions from either end resulted in the complete inactivation of the toxin. The study demonstrates that intact N- and C-termini are required for the optimum functional activity of restrictocin.  相似文献   

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