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1.
Katoh S Murata K Kubota Y Kumeta H Ogura K Inagaki F Asayama M Katoh E 《Protein expression and purification》2005,43(2):113-156
OsNifU1A is a NifU-like rice (Oryza sativa) protein, discovered recently. Its amino acid sequence is very homologous to the sequence of cyanobacterial CnfU and to the sequences of NifU C-terminal domains. Based on its sequence, OsNifU1A is probably a modular structure consisting of two CnfU-like domains, with domain I (formed by residues Leu73 to Gly153) and domain II (formed by residues Leu154 to Ser226). Domain I have a conserved Cys-X-X-Cys motif, which may function as an iron-sulfur cluster assembly scaffold. Domain II lacks a Cys-X-X-Cys motif and therefore, cannot function analogously. Other NifU-like proteins, with sequences homologous to OsNifU1A domain II, have been identified during plant genomic projects; however, the biological roles of these domains remain unknown. We successfully constructed an Escherichia coli expression system for OsNifU1A domain II that enabled us to synthesize and purify milligram quantities of protein for use in structural and functional studies. Using the Gateway system, we built DNA sequences corresponding to two OsNifU1A domain II fusion proteins. One construct has a (His)6 sequence upstream of the OsNifU1A domain II sequence; the other has an upstream thioredoxin-(His)6 sequence. Recombinant OsNifU1A domain II fusion proteins were extracted from E. coli inclusion bodies by dissolving them in 6 M guanidine-HCl. About 36% of the total (His)6/OsNifU1A domain II fusion protein initially present remained soluble after guanidine-HCl was completely removed by step-wise dialysis; whereas, recovery of soluble Trx-(His)6 fusion protein was about 60% of the total cell lysate. About 2 mg of 15N-labeled OsNifU1A domain II was purified for NMR spectral studies. Examination of the OsNifU1A domain II 1H-15N HSQC NMR spectrum indicated that the purified protein was monomeric and correctly folded. Therefore, we established an efficient procedure for synthesis and purification of 15N-labeled OsNifU1A domain II in quantities sufficient for heteronuclear NMR solution structure studies. 相似文献
2.
The cooperative role of OsCnfU-1A domain I and domain II in the iron sulphur cluster transfer process as revealed by NMR 总被引:1,自引:0,他引:1
Saio T Kumeta H Ogura K Yokochi M Asayama M Katoh S Katoh E Teshima K Inagaki F 《Journal of biochemistry》2007,142(1):113-121
OsCnfU-1A is a chloroplast-type Nfu-like protein that consists of tandem repeats sharing high sequence homology. Domain I of this protein, but not domain II, has a C-X-X-C motif that is thought to assemble an iron-sulphur cluster. Herein we report the solution structure of OsCnfU-1A domain I (73-153). Although OsCnfU-1A domain I is structurally similar to OsCnfU-1A domain II (154-226), the electrostatic surface potential of the 2 domains differs. Domain I has an acidic surface, whereas that of domain II is predominantly basic. Chemical shift perturbation studies on OsCnfU-1A domain I and domain II with ferredoxin revealed negligible chemical shift changes in domain I, whereas much larger chemical shift changes were observed in domain II. The residues with larger chemical shift changes were located on the basic surface of domain II. Considering that ferredoxin is predominantly negatively charged, we propose the following hypothesis: First, an iron-sulphur cluster is assembled on domain I. Next, domain II interacts with the ferredoxin, thus tethering domain I close to the ferredoxin. Finally, domain I transfers the iron-sulphur cluster to the ferredoxin. Thus, domain II facilitates the efficient transfer of the iron-sulphur cluster from domain I to the ferredoxin. 相似文献
3.
Assembly of the functional tetrameric form of Mu transposase (MuA protein) at the two att ends of Mu depends on interaction
of MuA with multiple att and enhancer sites on supercoiled DNA, and is stimulated by MuB protein. The N-terminal domain I
of MuA harbours distinct regions for interaction with the att ends and enhancer; the C-terminal domain III contains separate
regions essential for tetramer assembly and interaction with MuB protein (IIIα and IIIβ, respectively). Although the central
domain II (the ‘DDE’ domain) of MuA harbours the known catalytic DDE residues, a 26 amino acid peptide within IIIα also has
a non-specific DNA binding and nuclease activity which has been implicated in catalysis. One model proposes that active sites
for Mu transposition are assembled by sharing structural/catalytic residues between domains II and III present on separate
MuA monomers within the MuA tetramer. We have used substrates with altered att sites and mixtures of MuA proteins with either
wild-type or altered att DNA binding specificities, to create tetrameric arrangements wherein specific MuA subunits are nonfunctional
in II, IIIα or IIIβ domains. From the ability of these oriented tetramers to carry out DNA cleavage and strand transfer we
conclude that domain IIIα or IIIβ function is not unique to a specific subunit within the tetramer, indicative of a structural
rather than a catalytic function for domain III in Mu transposition. 相似文献
4.
Replacement of glycine 227 in the fifth WD40 motif of α-COP/Ret1p/Soo1p by charged or aromatic amino acids is responsible
for the temperature-dependent osmo-sensitivity of Saccharomyces cerevisiae, while truncations of WD40 motifs exerted a reduction in cell growth rate and impairment in assembly of cell-wall associated
proteins such as enolase and Gas1p. Yeast two-hybrid analysis revealed that the ret1-1/soo1-1 mutation of α-COP abolished the interaction with β- and ɛ-COP, respectively, and that the interaction between α-COP and β-COP relied on the WD40 domain of α-COP. Furthermore, although
the WD40 domain is dispensable for interaction of α-COP with ɛ-COP, structural alterations in the WD40 domain could impair the interaction. 相似文献
5.
The biosynthesis of iron-sulfur clusters is a highly regulated process involving several proteins. Among them, so-called scaffold proteins play pivotal roles in both the assembly and delivery of iron-sulfur clusters. Here, we report the identification of two chloroplast-localized NifU-like proteins, AtCnfU-V and AtCnfU-IVb, from Arabidopsis (Arabidopsis thaliana) with high sequence similarity to a cyanobacterial NifU-like protein that was proposed to serve as a molecular scaffold. AtCnfU-V is constitutively expressed in several tissues of Arabidopsis, whereas the expression of AtCnfU-IVb is prominent in the aerial parts. Mutant Arabidopsis lacking AtCnfU-V exhibited a dwarf phenotype with faint pale-green leaves and had drastically impaired photosystem I accumulation. Chloroplasts in the mutants also showed a decrease in both the amount of ferredoxin, a major electron carrier of the stroma that contains a [2Fe-2S] cluster, and in the in vitro activity of iron-sulfur cluster insertion into apo-ferredoxin. When expressed in Escherichia coli cells, AtCnfU-V formed a homodimer carrying a [2Fe-2S]-like cluster, and this cluster could be transferred to apo-ferredoxin in vitro to form holo-ferredoxin. We propose that AtCnfU has an important function as a molecular scaffold for iron-sulfur cluster biosynthesis in chloroplasts and thereby is required for biogenesis of ferredoxin and photosystem I. 相似文献
6.
Atsushi Ohshima Susumu Uchiyama Hiroaki Nakano Takuya Yoshida Tadayasu Ohkubo Yuji Kobayashi 《International journal of peptide research and therapeutics》2003,10(5-6):539-543
Summary During investigation of thermal transitions of peptides and proteins, the transition temperature is occasionally too high
to trace the whole transition profile. In order to solve this problem and perform conformational analysis at high temperature,
we have recently developed a pressure-proof cell compartment for circular dichroism measurements. Here we demonstrate how
well this system works to collect CD spectra, at high temperature up to 180°c in aqueous solution. Ribosome recycling factor
(RRF), which consists of two domains; three stranded α-helix bundle domain (Domain I) and β/α/β domain (Domain II), was used
as an example. We constructed models of isolated Domain I substituting Domain II with tripeptide (Gly-Gly-Gly) and compared
these models from mesophilic and thermophilic bacteria. The melting profiles of these models revealed that thermal stability
is enhanced by the increased enthalpy provided by hydrogen bonds and ionic pairing. 相似文献
7.
8.
Smith AD Jameson GN Dos Santos PC Agar JN Naik S Krebs C Frazzon J Dean DR Huynh BH Johnson MK 《Biochemistry》2005,44(39):12955-12969
NifU is a homodimeric modular protein comprising N- and C-terminal domains and a central domain with a redox-active [2Fe-2S](2+,+) cluster. It plays a crucial role as a scaffold protein for the assembly of the Fe-S clusters required for the maturation of nif-specific Fe-S proteins. In this work, the time course and products of in vitro NifS-mediated iron-sulfur cluster assembly on full-length NifU and truncated forms involving only the N-terminal domain or the central and C-terminal domains have been investigated using UV-vis absorption and M?ssbauer spectroscopies, coupled with analytical studies. The results demonstrate sequential assembly of labile [2Fe-2S](2+) and [4Fe-4S](2+) clusters in the U-type N-terminal scaffolding domain and the assembly of [4Fe-4S](2+) clusters in the Nfu-type C-terminal scaffolding domain. Both scaffolding domains of NifU are shown to be competent for in vitro maturation of nitrogenase component proteins, as evidenced by rapid transfer of [4Fe-4S](2+) clusters preassembled on either the N- or C-terminal domains to the apo nitrogenase Fe protein. Mutagenesis studies indicate that a conserved aspartate (Asp37) plays a critical role in mediating cluster transfer. The assembly and transfer of clusters on NifU are compared with results reported for U- and Nfu-type scaffold proteins, and the need for two functional Fe-S cluster scaffolding domains on NifU is discussed. 相似文献
9.
Frataxin is an iron-binding mitochondrial matrix protein that has been shown to mediate iron delivery during iron–sulfur cluster
and heme biosynthesis. Mitochondrial processing peptidase (MPP) yields a form of human frataxin corresponding to residues
56–210. However, structural and functional studies have focused on a core structure that results from an ill-defined cleavage
event at the N-terminus. Herein we show that the N-terminus of MPP-processed frataxin shows a unique high-affinity iron site
and that this iron center appears to mediate a self-cleavage reaction. Moreover, the N-terminus appears to block previously
defined iron-binding sites located on the carboxylate-rich surface defined by the helix (α1) and the β-sheet (β1), most likely
through electrostatic contact with the carboxylate-rich surface on the core protein, as well as inhibiting iron-promoted binding
of the iron–sulfur cluster assembly scaffold partner protein, ISU. The physiological significance of iron-mediated release
of the N-terminal residues from this anionic surface is discussed. 相似文献
10.
The interactions between negatively charged β-lactoglobulin and the positively charged lactoferrin at the droplet surface
to form a multi-protein surface layer were examined. Addition of lactoferrin to the aqueous phase of emulsions formed with
β-lactoglobulin at pH 7.0 caused an increase in the ζ-potential of emulsion droplets, and the ζ-potential became positive
as the concentration of added lactoferrin was higher than 1% in the system. It is found that lactoferrin binds to adsorbed
β-lactoglobulin at droplet surface probably via electrostatic interactions. The amount of lactoferrin at interface increased
with increasing the concentration of added lactoferrin, but it decreased with a decrease in the pH. No lactoferrin was observed
at interface at pH 3 and 4. By contrast, when β-lactoglobulin was added in the emulsions formed with lactoferrin at pH 7.0,
the ζ-potential of emulsions changed from positive to negative as the concentration of added β-lactoglobulin increased. The
amount of β-lactoglobulin at surface increased correspondingly with increasing the concentration of added β-lactoglobulin.
However, in this case, β-lactoglobulin remained bound at interface even at pH 3 and 4 where both lactoferrin and β-lactoglobulin
are positively charged. The association of lactoferrin or β-lactoglobulin with the surface proteins that have oppositely charge
is probably mainly through electrostatic interactions between the two proteins. It appears that alternative layers of these
proteins could be created at the droplet surface. 相似文献
11.
Osamu Takenaka Mika Hotta Akiko Takenaka Yoshi Kawamoto Bambang Suryobroto Edy Brotoisworo 《Primates; journal of primatology》1987,28(1):87-98
The monkeys on the island of Sulawesi (Celebes), Indonesia, comprise seven species ofMacaca, that isM. maura, M. tonkeana, M. hecki, M. nigrescens, M. nigra, M. ochreata, andM. brunnescens. Hemoglobins from 248 individuals of these seven species were analyzed by isoelectric focusing electrophoresis (IEFE) and
by starch gel electrophoresis in the presence of urea (USGE). Eighteen phenotypes consisting of eight molecular types were
identified by IEFE analysis. The speciestonkeana inhabiting the central part of the island revealed 11 phenotypes, while peripheral species such asnigrescens andbrunnescens carried only 3 and 2 phenotypes, respectively.
On USGE, three α chains and three β chains were identified and named α1, α2, and α6, and β1, β3, and β5, respectively. The
α1 chain has the same mobility as the α chains of other macaques, while the α2 chain is less positively charged than α1, and
α6 is the least positive among these α chains. The α2 chain is widely distributed in the Sulawesi macaques as the major component.
Four species,ochreata, tonkeana, maura, andnigrescens, carried the α1 and α6 chains as minor components. The electrophoretic mobility of β1 was the same as that of other macaques,
while β3 and β5 were more positively charged and less positively charged than β1, respectively. All of the Sulawesi species
had β3 in high or low gene frequencies and inmaura, tonkeana, andbrunnescens, this type was most abundant. β5 chain existed in the species of the northern peninsula, as the major type. The subordinate
type was β3 innigra andnigrescens and β1 inhecki. On the other hand, β1 was most frequently observed inochreata. 相似文献
12.
Integrins are cell adhesion receptors that are evolutionary old and that play important roles during developmental and pathological
processes. The integrin family is composed of 24 αβ heterodimeric members that mediate the attachment of cells to the extracellular
matrix (ECM) but that also take part in specialized cell-cell interactions. Only a subset of integrins (8 out of 24) recognizes
the RGD sequence in the native ligands. In some ECM molecules, such as collagen and certain laminin isoforms, the RGD sequences
are exposed upon denaturation or proteolytic cleavage, allowing cells to bind these ligands by using RGD-binding receptors.
Proteolytic cleavage of ECM proteins might also generate fragments with novel biological activity such as endostatin, tumstatin,
and endorepellin. Nine integrin chains contain an αI domain, including the collagen-binding integrins α1β1, α2β1, α10β1, and
α11β1. The collagen-binding integrins recognize the triple-helical GFOGER sequence in the major collagens, but their ability
to recognize these sequences in vivo is dependent on the fibrillar status and accessibility of the interactive domains in
the fibrillar collagens. The current review summarizes some basic facts about the integrin family including a historical perspective,
their structure, and their ligand-binding properties. 相似文献
13.
Rhodobacter capsulatus contains lhaA and pucC genes that have been implicated in light-harvesting complex 1 and 2 (LH1 and LH2) assembly. The proteins encoded by these
genes, and homologues in other photosynthetic organisms, have been classified as the bacteriochlorophyll delivery (BCD) family
of the major facilitator superfamily. A new BCD family phylogenetic tree reveals that several PucC, LhaA and Orf428-related
sequences each form separate clusters, while plant and cyanobacterial homologues cluster more distantly. The PucC protein
is encoded in the pucBACDE superoperon which also codes for LH2 α (PucA) and β (PucB) proteins. PucC was previously shown to be necessary for formation
of LH2. This article gives evidence indicating that PucC has a shepherding activity that keeps the homologous α and β proteins
of LH1 and LH2 apart, allowing LH1 to assemble properly. This shepherding function was indicated by a 62% reduction in LH1
levels in ΔLHII strains carrying plasmids encoding pucBA along with a C-terminally truncated pucC gene. More severe reductions in LH1 were seen when the truncated pucC gene was co-expressed in the presence of C-terminal PucC::PhoA fusion proteins. It appears that interaction between truncated
PucC::PhoA fusion proteins and the truncated PucC protein disrupts LH1 assembly, pointing towards a PucC dimeric or multimeric
functional unit. 相似文献
14.
Dos Santos PC Smith AD Frazzon J Cash VL Johnson MK Dean DR 《The Journal of biological chemistry》2004,279(19):19705-19711
The NifU protein is a homodimer that is proposed to provide a molecular scaffold for the assembly of [Fe-S] clusters uniquely destined for the maturation of the nitrogenase catalytic components. There are three domains contained within NifU, with the N-terminal domain exhibiting a high degree of primary sequence similarity to a related family of [Fe-S] cluster biosynthetic scaffolds designated IscU. The C-terminal domain of NifU exhibits sequence similarity to a second family of proposed [Fe-S] cluster biosynthetic scaffolds designated Nfu. Genetic experiments described here involving amino acid substitutions within the N-terminal and C-terminal domains of NifU indicate that both domains can separately participate in nitrogenase-specific [Fe-S] cluster formation, although the N-terminal domain appears to have the dominant function. These in vivo experiments were supported by in vitro [Fe-S] cluster assembly and transfer experiments involving the activation of an apo-form of the nitrogenase Fe protein. 相似文献
15.
Ningzhi Xu Omar Coso Daruka Mahadevan Antonio De Blasi Paul K. Goldsmith William F. Simonds J. Silvio Gutkind 《Cellular and molecular neurobiology》1996,16(1):51-59
Summary 1. The noncatalytic domain of Ras-GAP can affect signaling through G protein-coupled receptors by a poorly understood mechanism.
2. In this study, fusion proteins containing elements of the noncatalytic domain ofras-GAP were examined for their ability to bindβγ subunits of heterotrimeric G proteins and phosphotyrosine-containing polypeptides.
3. Our results demonstrate that purifiedβγ dimers associated with bacterially expressed GAP proteins and that this association does not require SH2 or SH3 domains but
is dependent on the presence of the GAP pleckstrin-homology (PH) domain. In contrast, only the SH2 domains are necessary for
binding to tyrosine phosphorylated proteins.
4. These findings raise the possibility that heterotrimeric G proteins might affect functioning ofras-like proteins throughβγ subunits acting on their regulatory molecules. 相似文献
16.
In a previous report (Cebo et al. J Biol Chem 276 (2001) 5685–5691), it was established that biologically active recombinant human IL-1α and IL-1β had different carbohydrate-binding
properties. IL-1α recognized a di-antennary N-glycan with two α2-3-linked sialic acid residues, whereas IL-1β recognized the
GM4, a α2-3-linked sialylated glycosphingolipid. These different carbohydrate-binding properties of two interleukins binding
to the same receptor (IL-1R) could explain why these molecules had different biological effects and cell specificities. Molecular
modeling of the ligands and in silico docking experiments defined putative carbohydrate-recognition domains localized in the same area of the two molecules, a
domain different from that defined as the type I IL-1R binding domain. The calculated pattern of hydrogen bonding and of van
der Waals interactions fulfilled the essential features observed for calcium-independent lectins (mammalian, viral or bacterial).
The analysis of the same domain of the third members of this family of molecules, the IL-1R-antagonist, indicated it did not
fulfill the criteria for carbohydrate-recognition domains. It is proposed that its role as a pure antagonist is due to the
absence of lectin activity and consequently explained its inability to associate IL-1R with other surface molecular complexes
necessary for signaling. 相似文献
17.
Indolepyruvate ferredoxin oxidoreductase (IOR) catalyzes the oxidative decarboxylation of arylpyruvates. Gene cloning and
sequencing analysis of the IOR gene from the hyperthermophilic archaeon Pyrococcus sp. KOD1 was performed. Two genes, iorA and iorB, encoding α and β subunits of IOR were found to be tandemly arranged, which suggests that gene expression is translationaly
coupled. Sequence analysis showed the C-terminal region of the α subunit to have a typical ferredoxin-type [4Fe-4S] cluster
motif (CXXCXXCXXCXXXCP), which is similar to that present in the δ subunits of other oxidoreductases such as pyruvate ferredoxin
oxidoreductase (POR) and 2-ketoisovalerate ferredoxin oxidoreductase (VOR). We suggest that the α subunit of KOD1-IOR has
a mosaic structure composed of features characteristic of the α, β and δ subunits from POR and VOR. KOD1-IOR was overproduced
in anaerobically incubated Escherichia coli cells and the crude enzyme was extracted under anaerobic conditions. The optimal temperature for activity of recombinant
IOR was 70° C and the half-life of this enzyme in the presence of air was 15 min at 25° C.
Received: 25 September 1996 / Accepted: 20 December 1996 相似文献
18.
Protein domain frequency and distribution among kingdoms was statistically analyzed using the SCOP structural database. It
appeared that among chosen protein domains with the best resolution, eukaryotic proteins more often belong to α-helical and
β-structural proteins, while proteins of bacterial origin belong to α/β structural class. Statistical analysis of folding
rates of 73 proteins with known experimental data revealed that bacterial proteins with simple kinetics (23 proteins) exhibit
a higher folding rate compared to eukaryotic proteins with simple folding kinetics (27 proteins). Analysis of protein domain
amino acid composition showed that the frequency of amino acid residues in proteins of eukaryotic and bacterial origin is
different for proteins with simple and complex folding kinetics. 相似文献
19.
Zhou Qinwei Desai Smruti A. Wang Xinhui Noronha Elvyra J. Neri Mariangela Ferrone Soldano 《International journal of peptide research and therapeutics》1999,6(1):77-86
Summary Utilizing phage display peptide libraries, we have identified and mapped the antigenic determinants recognized by mouse monoclonal
antibodies (mAb) on two sets of immunologically important molecules, HLA class I and class II antigens. Anti-HLA class I mAb
TP25.99 recognizes a conformational and a linear determinant on distinct regions of the HLA class I α3 domain. Anti-HLA class
I mAb HO-4 recognizes a conformational determinant on the α2 domain of HLA-A2 and A28 allospecificities. Anti-HLA-DR1,-DR4,-DR6,-DR8,-DR9
mAb SM/549 recognizes a conformational determinant on the β chain of HLA class II antigens. These results indicate the versatility
of phage display peptide libraries to characterize antigenic determinants recognized by anti-HLA mAb. 相似文献
20.
Eletsky A Acton TB Xiao R Everett JK Montelione GT Szyperski T 《Journal of structural and functional genomics》2012,13(1):9-14
The protein family (Pfam) PF04536 is a broadly conserved domain family of unknown function (DUF477), with more than 1,350
members in prokaryotic and eukaryotic proteins. High-quality NMR structures of the N-terminal domain comprising residues 41–180
of the 684-residue protein CG2496 from Corynebacterium glutamicum and the N-terminal domain comprising residues 35–182 of the 435-residue protein PG0361 from Porphyromonas gingivalis both exhibit an α/β fold comprised of a four-stranded β-sheet, three α-helices packed against one side of the sheet, and
a fourth α-helix attached to the other side. In spite of low sequence similarity (18%) assessed by structure-based sequence
alignment, the two structures are globally quite similar. However, moderate structural differences are observed for the relative
orientation of two of the four helices. Comparison with known protein structures reveals that the α/β architecture of CG2496(41–180)
and PG0361(35–182) has previously not been characterized. Moreover, calculation of surface charge potential and identification
of surface clefts indicate that the two domains very likely have different functions. 相似文献