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1.
In the ancient organisms, methanogenic archaea, lacking the canonical cysteinyl-tRNA synthetase, Cys-tRNA(Cys) is produced by an indirect pathway, in which O-phosphoseryl-tRNA synthetase ligates O-phosphoserine (Sep) to tRNA(Cys) and Sep-tRNA:Cys-tRNA synthase (SepCysS) converts Sep-tRNA(Cys) to Cys-tRNA(Cys). In this study, the crystal structure of SepCysS from Archaeoglobus fulgidus has been determined at 2.4 A resolution. SepCysS forms a dimer, composed of monomers bearing large and small domains. The large domain harbors the seven-stranded beta-sheet, which is typical of the pyridoxal 5'-phosphate (PLP)-dependent enzymes. In the active site, which is located near the dimer interface, PLP is covalently bound to the side-chain of the conserved Lys209. In the proximity of PLP, a sulfate ion is bound by the side-chains of the conserved Arg79, His103, and Tyr104 residues. The active site is located deep within the large, basic cleft to accommodate Sep-tRNA(Cys). On the basis of the surface electrostatic potential, the amino acid residue conservation mapping, the position of the bound sulfate ion, and the substrate amino acid binding manner in other PLP-dependent enzymes, a binding model of Sep-tRNA(Cys) to SepCysS was constructed. One of the three strictly conserved Cys residues (Cys39, Cys42, or Cys247), of one subunit may play a crucial role in the catalysis in the active site of the other subunit. 相似文献
2.
Liu Y Dos Santos PC Zhu X Orlando R Dean DR Söll D Yuan J 《The Journal of biological chemistry》2012,287(8):5426-5433
Sep-tRNA:Cys-tRNA synthase (SepCysS) catalyzes the sulfhydrylation of tRNA-bound O-phosphoserine (Sep) to form cysteinyl-tRNA(Cys) (Cys-tRNA(Cys)) in methanogens that lack the canonical cysteinyl-tRNA synthetase (CysRS). A crystal structure of the Archaeoglobus fulgidus SepCysS apoenzyme provides information on the binding of the pyridoxal phosphate cofactor as well as on amino acid residues that may be involved in substrate binding. However, the mechanism of sulfur transfer to form cysteine was not known. Using an in vivo Escherichia coli complementation assay, we showed that all three highly conserved Cys residues in SepCysS (Cys(64), Cys(67), and Cys(272) in the Methanocaldococcus jannaschii enzyme) are essential for the sulfhydrylation reaction in vivo. Biochemical and mass spectrometric analysis demonstrated that Cys(64) and Cys(67) form a disulfide linkage and carry a sulfane sulfur in a portion of the enzyme. These results suggest that a persulfide group (containing a sulfane sulfur) is the proximal sulfur donor for cysteine biosynthesis. The presence of Cys(272) increased the amount of sulfane sulfur in SepCysS by 3-fold, suggesting that this Cys residue facilitates the generation of the persulfide group. Based upon these findings, we propose for SepCysS a sulfur relay mechanism that recruits both disulfide and persulfide intermediates. 相似文献
3.
Structure-based mutagenesis was used to probe the binding surface for the activation domain of sterol-responsive element binding protein (SREBP) in the KIX domain of CREB binding protein. A set of conserved residues scattering in the alpha2 helix and the extended C-terminal region of alpha 3 helix in the KIX domain including two arginines previously characterized as a hot spot for cofactor-mediated methylation was shown to be crucial for SREBP-KIX interaction, and was not essential for phosphorylated KID recognition. Therefore, our results suggest the existence of a SREBP binding site formed by positively charged residues in the C-terminal part of the extended alpha 3 helix of the KIX domain distinct from the previously identified phosphorylated KID binding site. 相似文献
4.
Bruno Ramos-Molina Ana LambertosAndrés J. López-Contreras Rafael Peñafiel 《Biochimica et Biophysica Acta (BBA)/General Subjects》2013
Background
Ornithine decarboxylase (ODC), the key enzyme in the polyamine biosynthetic pathway, is highly regulated by antizymes (AZs), small proteins that bind and inhibit ODC and increase its proteasomal degradation. Early studies delimited the putative AZ-binding element (AZBE) to the region 117-140 of ODC. The aim of the present work was to study the importance of certain residues of the region 110-142 that includes the AZBE region for the interaction between ODC and AZ1 and the ODC functionality.Methods
Computational analysis of the protein sequences of the extended AZBE site of ODC and ODC paralogues from different eukaryotes was used to search for conserved residues. The influence of these residues on ODC functionality was studied by site directed mutagenesis, followed by different biochemical techniques.Results
The results revealed that: a) there are five conserved residues in ODC and its paralogues: K115, A123, E138, L139 and K141; b) among these, L139 is the most critical one for the interaction with AZs, since its substitution decreases the affinity of the mutant protein towards AZs; c) all these conserved residues, with the exception of A123, are critical for ODC activity; d) substitutions of K115, E138 or L139 diminish the formation of ODC homodimers.Conclusions
These results reveal that four of the invariant residues of the AZBE region are strongly related to ODC functionality.General significance
This work helps to understand the interaction between ODC and AZ1, and describes various new residues involved in ODC activity, a key enzyme for cell growth and proliferation. 相似文献5.
CTPS (cytidine 5'-triphosphate synthase) catalyses the ATP-dependent formation of CTP from UTP using either ammonia or L-glutamine as the nitrogen source. Binding of the substrates ATP and UTP, or the product CTP, promotes oligomerization of CTPS from inactive dimers to active tetramers. In the present study, site-directed mutagenesis was used to replace the fully conserved glycine residues 142 and 143 within the UTP-binding site and 146 within the CTP-binding site of Escherchia coli CTPS. CD spectral analyses of wild-type CTPS and the glycine mutants showed a slight reduction of approximately 15% in alpha-helical content for G142A and G143A relative to G146A and wild-type CTPS, suggesting some local alterations in structure. Relative to wild-type CTPS, the values of k(cat)/K(m) for ammonia-dependent and glutamine-dependent CTP formation catalysed by G143A were reduced 22- and 16-fold respectively, whereas the corresponding values for G146A were reduced only 1.4- and 1.8-fold respectively. The glutaminase activity (k(cat)) of G146A was similar to that exhibited by the wild-type enzyme, whereas that of G143A was reduced 7.5-fold. G146A exhibited substrate inhibition at high concentrations of ammonia and a partial uncoupling of glutamine hydrolysis from CTP production. Although the apparent affinity (1/[S](0.5)) of G143A and G146A for UTP was reduced approximately 4-fold, G146A exhibited increased co-operativity with respect to UTP. Thus mutations in the CTP-binding site can affect UTP-dependent activity. Surprisingly, G142A was inactive with both ammonia and glutamine as substrates. Gel-filtration HPLC experiments revealed that both G143A and G146A were able to form active tetramers in the presence of ATP and UTP; however, nucleotide-dependent tetramerization of G142A was significantly impaired. Our observations highlight the sensitivity of the structure of CTPS to mutations in the UTP- and CTP-binding sites, with Gly(142) being critical for nucleotide-dependent oligomerization of CTPS to active tetramers. This 'structural sensitivity' may limit the number and/or types of mutations that could be selected for during the development of resistance to cytotoxic pyrimidine nucleotide analogues. 相似文献
6.
Background
The mini-chromosome maintenance protein (MCM) complex is an essential replicative helicase for DNA replication in Archaea and Eukaryotes. While the eukaryotic complex consists of six homologous proteins (MCM2-7), the archaeon Sulfolobus solfataricus has only one MCM protein (ssoMCM), six subunits of which form a homohexamer. We have recently reported a 4.35Å crystal structure of the near full-length ssoMCM. The structure reveals a total of four β-hairpins per subunit, three of which are located within the main channel or side channels of the ssoMCM hexamer model generated based on the symmetry of the N-terminal Methanothermobacter thermautotrophicus (mtMCM) structure. The fourth β-hairpin, however, is located on the exterior of the hexamer, near the exit of the putative side channels and next to the ATP binding pocket.Results
In order to better understand this hairpin's role in DNA binding and helicase activity, we performed a detailed mutational and biochemical analysis of nine residues on this exterior β-hairpin (EXT-hp). We examined the activities of the mutants related to their helicase function, including hexamerization, ATPase, DNA binding and helicase activities. The assays showed that some of the residues on this EXT-hp play a role for DNA binding as well as for helicase activity.Conclusions
These results implicate several current theories regarding helicase activity by this critical hexameric enzyme. As the data suggest that EXT-hp is involved in DNA binding, the results reported here imply that the EXT-hp located near the exterior exit of the side channels may play a role in contacting DNA substrate in a manner that affects DNA unwinding.7.
Chalcone synthase (CHS), a key enzyme in flavonoid biosynthesis, catalyses sequential decarboxylative condensations of p-coumaroyl-CoA with three malonyl-CoA molecules and cyclizes the resulting tetraketide intermediate to produce chalcone. Phenylglyoxal, an Arg selective reagent, was found to inactivate the enzyme, although no Arg is found at the active site. Conserved, non-active site Arg residues of CHS were individually mutated and the results were discussed in the context of the 3D structure of CHS. Arg199 and Arg350 were shown to provide important interactions to maintain the structural integrity and foldability of the enzyme. Arg68, Arg172 and Arg328 interact with highly conserved Gln33/Phe215, Glu380 and Asp311/Glu314, respectively, thus helping position the catalytic Cys-His-Asn triad and the (372)GFGPG loop in correct topology at the active site. In particular, a mutation of Arg172 resulted in selective impairment in the cyclization activities of CHS and stilbene synthase, a related enzyme that catalyses a different cyclization of the same tetraketide intermediate. These Arg residues and their interactions are well conserved in other enzymes of the CHS superfamily, suggesting that they may serve similar functions in other enzymes. Mutations of Arg68 and Arg328 had been found in mutant plants that showed impaired CHS activity. 相似文献
8.
Kex2/subtilisin-like proteinase activity is required for the production of the adult cuticle in the nematode Caenorhabditis elegans. Deletion of the carboxy termini of four of the bli-4/kpc-4 convertase isoforms results in blistering of the adult cuticle. The blisters vary in severity (expressivity) and are not evident in all individuals (reduced penetrance). We have isolated 13 bli-4/kpc-4 mutants that arrest development in late embryogenesis. Using a PCR-based heteroduplex technique, we have identified nucleotide changes responsible for eight of these lethal mutations. The lesions reside within the first 12 exons that are shared by all of the bli-4/kpc-4 gene products, with the majority of mutations clustered within the protease domain. This finding suggests that the protease domain represents a large mutable target. Among these mutations, allele h384 represents a molecular null mutant in which the catalytically essential serine residue (Ser415) is replaced by phenylalanine. Novel missense mutations that change the identity of amino acids evolutionary conserved in all kex2/subtilisin-convertases highlight critical residues essential for activity. We examined the functional activity of BLI-4/KPC-4 products expressed from several lethal mutants by testing their effect on the variable penetrance of blistering exhibited by the e937 allele. We found that the combination of a bli-4/kpc-4 lethal mutation in trans to the bli-4(e937) mutation was sufficient to cause severe blistering in heteroallelic progeny, even in the presence of a known dominant suppressor. 相似文献
9.
Terada T Fujita N Adachi H Nanjo H Sato R Takagi T Maeda M 《Biochimica et biophysica acta》2001,1547(1):127-134
The cloning, bacterial expression and purification of bovine liver cytosolic dihydrodiol dehydrogenase 3 (DD3) cDNA (1330 bp in full length) using the pKK223-3 expression vector has been reported previously. Recombinant DD3 (rDD3) was characterized in terms of its substrate specificity and inhibitor sensitivity [Terada et al., Adv. Exp. Biol. Res. 414 (1997) 543-553]. The nucleotide sequence of DD3 cDNA completely matched with that of bovine liver-type prostaglandin F synthase [Suzuki et al., J. Biol. Chem. 274 (1999) 241-248]. In the present study, we succeeded in high level expression of rDD3 in Escherichia coli BL21 (DE3) using the pET28a expression vector. rDD3 was easily and quickly purified to apparent homogeneity by one-step column chromatography using Ni(2+)-affinity resin. Furthermore, rDD3 showed essentially the same substrate specificity and inhibitor sensitivity to that of purified liver DD3. To analyze the role of cysteines (145, 154, 188, 193 and 206) in the enzymatic activity of DD3, site-directed mutagenesis of DD3 using the polymerase chain reaction method was performed. Mutants (C145S, C154S, C188S, C193S and C206S) were analyzed for substrate specificity, cofactor binding and inactivation by disulfide (dithio-bis(2-nitrobenzoic acid), alkylating reagent (N-ethylmaleimide) and oxidants (naphthoquinone and H(2)O(2)) Results indicated that these five cysteines of rDD3 may not be directly involved in substrate or cofactor binding. Mutant C193S showed strong resistance to SH-reagents unlike wild-type DD3 (WT) or other mutants. Both the WT and the other mutants showed essentially the same sensitivity to SH-reagents. Cofactor (NADP(+)) protected mutants C145S, C188S and C206S from inactivation as well as WT, while NAD(+) was not protective. Our present results indicate that Cys193, which is located close to the NADP(+)-binding site, may be involved in the alteration of enzymatic activity. 相似文献
10.
Plummer LJ Hildebrandt ER Porter SB Rogers VA McCracken J Schmidt WK 《The Journal of biological chemistry》2006,281(8):4596-4605
The Ras converting enzyme (RCE) promotes a proteolytic activity that is required for the maturation of Ras, the yeast a-factor mating pheromone, and certain other proteins whose precursors bear a C-terminal CAAX tetrapeptide motif. Despite the physiological importance of RCE, the enzymatic mechanism of this protease remains undefined. In this study, we have evaluated the substrate specificity of RCE orthologs from yeast (Rce1p), worm, plant, and human and have determined the importance of conserved residues toward enzymatic activity. Our findings indicate that RCE orthologs have conserved substrate specificity, cleaving CVIA, CTLM, and certain other CAAX motifs, but not the CASQ motif, when these motifs are placed in the context of the yeast a-factor precursor. Our mutational studies of residues conserved between the orthologs indicate that an alanine substitution at His194 completely inactivates yeast Rce1p enzymatic activity, whereas a substitution at Glu156 or His248 results in marginal activity. We have also determined that residues Glu157, Tyr160, Phe190, and Asn252 impact the substrate selectivity of Rce1p. Computational methods predict that residues influencing Rce1p function are all near or within hydrophobic segments. Combined, our data indicate that yeast Rce1p function requires residues that are invariably conserved among an extended family of prokaryotic and eukaryotic enzymes and that these residues are likely to lie within or immediately adjacent to the transmembrane segments of this membrane-localized enzyme. 相似文献
11.
Mutational analysis of the cysteine residues in the hepatitis B virus small envelope protein. 总被引:13,自引:4,他引:9
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The small envelope protein of hepatitis B virus is the major component of the viral coat and is also secreted from cells as a 20-nm subviral particle, even in the absence of other viral proteins. Such empty envelope particles are composed of approximately 100 copies of this polypeptide and host-derived lipids and are stabilized by extensive intermolecular disulfide cross-linking. To study the contribution of disulfide bonds to assembly and secretion of the viral envelope, single and multiple mutants involving all 14 cysteines in HepG2 and COS-7 cells were analyzed. Of the six cysteines located outside the region carrying the surface antigen, Cys-48, Cys-65, and Cys-69 were each found to be essential for secretion of 20-nm particles, whereas Cys-76, Cys-90, and Cys-221 were dispensable. By introduction of an additional cysteine substituting serine 58, the yield of secreted particles was increased. Of four mutants involving the eight cysteines located in the antigenic region, only the double mutant lacking Cys-121 and Cys-124 was secreted with wild-type efficiency. Secretion-competent envelope proteins were intracellularly retained by secretion-deficient cysteine mutants. According to alkylation studies, both intracellular and secreted envelope proteins contained free sulfhydryl groups. Disulfide-linked oligomers were studied by gel electrophoresis under nonreducing conditions. 相似文献
12.
Mutational analysis of the Vibrio fischeri LuxI polypeptide: critical regions of an autoinducer synthase. 总被引:2,自引:0,他引:2
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B L Hanzelka A M Stevens M R Parsek T J Crone E P Greenberg 《Journal of bacteriology》1997,179(15):4882-4887
Synthesis of the Vibrio fischeri autoinducer, a signal involved in the cell density-dependent activation of bioluminescence, is directed by the luxI gene product. The LuxI protein catalyzes the synthesis of N-acyl-homoserine lactones from S-adenosylmethionine and acylated-acyl carrier protein. We have gained an appreciation of the LuxI regions and amino acid residues involved in autoinducer synthesis by isolating and analyzing mutations generated by random and site-specific mutagenesis of luxI. By random mutagenesis we isolated 13 different single amino acid substitutions in the LuxI polypeptide. Eleven of these substitutions resulted in no detectable autoinducer synthase activity, while the remaining two amino acid substitutions resulted in reduced but detectable activity. The substitutions that resulted in no detectable autoinducer synthase activity mapped to two small regions of LuxI. In Escherichia coli, wild-type luxI showed dominance over all of the mutations. Because autoinducer synthesis has been proposed to involve formation of a covalent bond between an acyl group and an active-site cysteine, we constructed site-directed mutations that altered each of the three cysteine residues in LuxI. All of the cysteine mutants retained substantial activity as an autoinducer synthase in E. coli. Based on the analysis of random mutations we propose a model in which there are two critical regions of LuxI, at least one of which is an intimate part of an active site, and based on the analysis of site-directed mutations we conclude that an active-site cysteine is not essential for autoinducer synthase activity. 相似文献
13.
Evaluation of critical structural elements of UDP-sugar substrates and certain cysteine residues of a vertebrate hyaluronan synthase 总被引:3,自引:0,他引:3
The hyaluronan (HA) synthases catalyze the addition of two different monosaccharides from UDP-sugar substrates to the linear heteropolysaccharide chain. To accomplish this task, the HA synthases must be able to bind and to transfer from both UDP-sugar substrates. Until now, it has been impossible to distinguish between these two abilities. We have created a mutant of xlHAS1, a HA synthase from Xenopus laevis, that allows for the examination of the enzyme's ability to bind substrate only. The ability of different compounds to protect the xlHAS1(C337S) mutant enzyme from loss of activity due to treatment with N-ethylmaleimide, a cysteine-modifying reagent, yields information on the relative affinity of a variety of nucleotides and nucleotide-sugars. We have observed that the substrate binding selectivity is more relaxed than the specificity of catalytic transfer. The only attribute that appears to be absolutely required for binding is a nucleotide containing two phosphates complexed with magnesium ion. The role of certain cysteine residues in catalysis was also evaluated. Cys307 of xlHAS1 may play a role in catalysis or in maintaining structure. Mutation of Cys337 raises the UDP-GlcUA Michaelis constant (K(m)), suggesting that this residue participates in UDP-GlcUA substrate binding or in catalytic complex formation. 相似文献
14.
15.
Inwardly rectifying potassium channels are important in cellular repolarization of many excitable tissues. Amino acid sequence alignment of different mammalian inward rectifier K(+) channels revealed two absolutely conserved cysteine residues in the putative extracellular face, suggesting a possible disulfide bond. Replacement of these cysteine residues in the Kir2.1 channel (i.e., C122 and C154) with either alanine or serine abolished current in Xenopus laevis oocytes although Western blotting established that the channels were fully expressed. The digestion pattern of channels treated with V8 protease combined with Western blotting under reducing and nonreducing conditions confirmed intrasubunit cross-linking of C122 and C154. Whole-cell and single channel current recordings of oocytes expressing tandem tetrameric constructs with one or two of the mutant subunits suggested that insertion of one mutant subunit is sufficient to eliminate channel function. Coexpression studies confirmed that the cysteine mutant channels eliminate wild-type Kir2.1 currents in a dominant-negative manner. Despite these results, sulfhydryl reduction did not alter the functional properties of Kir2.1 currents. Molecular modeling of Kir2.1 with the two cysteines cross-linked predicted that the extracellular loop between the first transmembrane domain and the pore helix contains a beta-hairpin structure. Distinct from the KcsA structure, the disulfide bond together with the beta-hairpin structure is expected to constrain and stabilize the P-loop and selectivity filter. Taken together, these results suggest that intramolecular disulfide bond exists between C122 and C154 of Kir2.1 channel and this cross-link might be required for proper channel folding. 相似文献
16.
Guarino LA Bhardwaj K Dong W Sun J Holzenburg A Kao C 《Journal of molecular biology》2005,353(5):1106-1117
The severe acute respiratory syndrome (SARS) coronavirus virus non-structural protein 15 is a Mn2+-dependent endoribonuclease with specificity for cleavage at uridylate residues. To better understand structural and functional characteristics of Nsp15, 22 mutant versions of Nsp15 were produced in Escherichia coli as His-tagged proteins and purified by metal-affinity and ion-exchange chromatography. Nineteen of the mutants were soluble and were analyzed for enzymatic activity. Six mutants, including four at the putative active site, were significantly reduced in endoribonuclease activity. Two of the inactive mutants had unusual secondary structures compared to the wild-type protein, as measured by circular dichroism spectroscopy. Gel-filtration analysis, velocity sedimentation ultracentrifugation, and native gradient pore electrophoresis all showed that the wild-type protein exists in an equilibrium between hexamers and monomers in solution, with hexamers dominating at micromolar protein concentration, while native gradient pore electrophoresis also revealed the presence of trimers. A mutant in the N terminus of Nsp15 was impaired in hexamer formation and had low endoribonuclease activity, suggesting that oligomerization is required for endoribonuclease activity. This idea was supported by titration experiments showing that enzyme activity was strongly concentration-dependent, indicating that oligomeric Nsp15 is the active form. Three-dimensional reconstruction of negatively stained single particles of Nsp15 viewed by transmission electron microscopic analysis suggested that the six subunits were arranged as a dimer of trimers with a number of cavities or channels that may constitute RNA binding sites. 相似文献
17.
18.
Mutational analysis of the adeno-associated virus Rep68 protein: identification of critical residues necessary for site-specific endonuclease activity. 总被引:1,自引:3,他引:1
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The Rep68 and Rep78 proteins of adeno-associated virus type 2 (AAV) are multifunctional proteins which contain overlapping amino acid sequences. They are required for viral replication and preferential integration of the AAV genome into a region of human chromosome 19. During the terminal resolution process of AAV DNA replication, these proteins make a site-specific and strand-specific endonuclease cut within the AAV inverted terminal repeat DNA. The Rep68 and Rep78 proteins also have helicase and DNA-binding activities. The endonuclease activity is believed to involve the covalent attachment of Rep68 or Rep78 at the cut site via a phosphotyrosine linkage. In an attempt to identify the active-site tyrosine residue of Rep78 and Rep68, tyrosine residues were site specifically mutated to phenylalanines by overlap extension PCR, and the resulting PCR fragments were cloned into a maltose binding protein-Rep68 fusion (MBP-Rep68delta) expression vector. The mutant MBP-Rep68delta proteins were expressed in Escherichia coli cells, purified with amylose resin, and assayed in vitro for Rep68-specific activities. Although several of the mutations disrupted the endonuclease activity, only the mutation of tyrosine 152 abrogated the endonuclease activity with no discernible effect on the helicase or DNA-binding activities. Our data therefore suggest that there are distinct active sites for the helicase and endonuclease activities. 相似文献
19.
Nicotinic acetylcholine receptors (nAChR) containing alpha7 subunits self-assemble into simple, homopentameric complexes. However, successful heterologous expression of functional alpha7-nAChR has only been achieved in a few host cell types, such as the SH-EP1 human epithelial cell line. All ionotropic glycine receptor, GABA(A) receptor, 5-HT(3) receptor, and nAChR subunits contain a pair of highly conserved cysteine residues (C150 and C164 for alpha7 subunits) in their N-terminal extracellular domain. These residues are thought to be involved in the formation of a conserved cystine loop that is critical to the proper folding and assembly of subunits. However, nAChR alpha7 (and alpha8) subunits also contain a third cysteine residue, C138, N-terminal to the conserved cysteine pair. Using SH-EP1 cells as a host for heterologous expression, we evaluated the roles of C138, C150, and C164 in subunit folding, assembly, and cell surface expression and function of alpha7-nAChR. Results indicate that mutation of C138, but not of C150 or C164, yields an nAChR that can assemble to form (125)I-labeled alpha-bungarotoxin binding sites expressed on the cell surface. Further, whole-cell patch clamp recordings demonstrate that mutation of C138 to alanine does not alter the function of the fully assembled alpha7-nAChR. These results indicate that C150 and C164 are required for surface expression, but that C138 is neither necessary for nor inhibitory toward the surface expression and function of human alpha7-nAChR. These results suggest that disulfide bond formation between C138 and either C150 or C164, if it occurs, has no significant effect on alpha7-nAChR assembly or function. 相似文献