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1.
    
CTP synthase catalyzes the last committed step in de novo pyrimidine‐nucleotide biosynthesis. Active CTP synthase is a tetrameric enzyme composed of a dimer of dimers. The tetramer is favoured in the presence of the substrate nucleotides ATP and UTP; when saturated with nucleotide, the tetramer completely dominates the oligomeric state of the enzyme. Furthermore, phosphorylation has been shown to regulate the oligomeric states of the enzymes from yeast and human. The crystal structure of a dimeric form of CTP synthase from Sulfolobus solfataricus has been determined at 2.5 Å resolution. A comparison of the dimeric interface with the intermolecular interfaces in the tetrameric structures of Thermus thermophilus CTP synthase and Escherichia coli CTP synthase shows that the dimeric interfaces are almost identical in the three systems. Residues that are involved in the tetramerization of S. solfataricus CTP synthase according to a structural alignment with the E. coli enzyme all have large thermal parameters in the dimeric form. Furthermore, they are seen to undergo substantial movement upon tetramerization.  相似文献   

2.
    
Phosphorylated derivatives of phosphatidylinositol (PtdIns), also called phosphoinositides (PIPs), are basic components of membrane‐associated signalling systems. A family of PtdIns‐transfer proteins (PITPs) called the Sec14 family have been predicted to form a set of functional modules that can sense different types of lipid metabolism and transmit the information to the PIP signalling system. In eukaryotic cells, the Sec14 family exhibits a wide diversity of activity, but the structural basis of this diversity remains unclear. In the present study, the dimeric structure of Sfh3 (Sec14 family homologue 3 in yeast) is reported for the first time and differs from the Sec14 proteins reported to date, all of which are monomeric. Some variations in the binding pocket of Sfh3 were observed and the dimer interface was identified and proposed to provide a link between dimer–monomer state changes and PtdIns binding. Together, these structural changes and the oligomeric state transformation of Sfh3 support ideas of diversity within the Sec14 family and provide some new clues to function.  相似文献   

3.
    
Human thymidylate synthase (hTS) provides the sole de novo intracellular source of thymidine 5′‐monophosphate (dTMP). hTS is required for DNA replication prior to cell division, making it an attractive target for anticancer chemotherapy and drug discovery. hTS binds 2′‐deoxyuridine 5′‐monophosphate (dUMP) and the folate co‐substrate N 5,N10‐methylenetetrahydrofolate (meTHF) in a pocket near the catalytic residue Cys195. The catalytic loop, which is composed of amino‐acid residues 181–197, can adopt two distinct conformations related by a 180° rotation. In the active conformation Cys195 is close to the active site, while in the inactive conformation it is rotated and Cys195 is too distant from the active site for catalysis. Several hTS structures, either native or engineered, have been solved in the active conformation in complex with ligands or inhibitors and at different salt concentrations. However, apo hTS structures have been solved in an inactive conformation in high‐salt and low‐salt conditions (PDB entries 1ypv , 4h1i , 4gyh , 3egy and 3ehi ). Here, the structure of apo hTS crystallized in the active form with sulfate ions coordinated by the arginine residue that binds dUMP is reported.  相似文献   

4.
    
In the last decade, advances in instrumentation and software development have made crystallography a powerful tool in structural biology. Using this method, structural information can now be acquired from pathological crystals that would have been abandoned in earlier times. In this paper, the order–disorder (OD) structure of fluorescent protein FP480 is discussed. The structure is composed of tetramers with 222 symmetry incorporated into the lattice in two different ways, namely rotated 90° with respect to each other around the crystal c axis, with tetramer axes coincident with crystallographic twofold axes. The random distribution of alternatively oriented tetramers in the crystal creates a rotational OD structure with statistically averaged I422 symmetry, although the presence of very weak and diffuse additional reflections suggests that the randomness is only approximate.  相似文献   

5.
    
Atomically dispersed Fe–N–C catalysts are considered the most promising precious‐metal‐free alternative to state‐of‐the‐art Pt‐based oxygen reduction electrocatalysts for proton‐exchange membrane fuel cells. The exceptional progress in the field of research in the last ≈30 years is currently limited by the moderate active site density that can be obtained. Behind this stands the dilemma of metastability of the desired FeN4 sites at the high temperatures that are believed to be a requirement for their formation. It is herein shown that Zn2+ ions can be utilized in the novel concept of active‐site imprinting based on a pyrolytic template ion reaction throughout the formation of nitrogen‐doped carbons. As obtained atomically dispersed Zn–N–Cs comprising ZnN4 sites as well as metal‐free N4 sites can be utilized for the coordination of Fe2+ and Fe3+ ions to form atomically dispersed Fe–N–C with Fe loadings as high as 3.12 wt%. The Fe–N–Cs are active electocatalysts for the oxygen reduction reaction in acidic media with an onset potential of E0 = 0.85 V versus RHE in 0.1 m HClO4. Identical location atomic resolution transmission electron microscopy imaging, as well as in situ electrochemical flow cell coupled to inductively coupled plasma mass spectrometry measurements, is employed to directly prove the concept of the active‐site imprinting approach.  相似文献   

6.
Three fluorescent probes were synthesized for studying the excitation energy migration between two identical fluorophores. Each probe has two identical fluorescent groups (dansyl, 7-nitrobenzo-2-oxa-1,3-diazole-4-yl, or fluoresceinyl) linked by the rigid bis-(8-aminooctyl)amide of 4,4"-biphenyldicarbonic acid or flexible dotriacontanedioic acid spacer, which enables the intramolecular energy migration through the distance of 3.2–3.5 nm.  相似文献   

7.
    
Three Rhodamine B derivatives were synthesized and characterized by ESI‐MS, NMR, HR‐MS and IR. The probes exhibit high selectivity and sensitivity towards Fe3+ over other metal ions in CH3CN–water. Upon the addition of Fe3+, the spirocyclic ring of the probe was opened and a significant enhancement of visible color and fluorescence within the range of 540–700 nm was observed. The colorimetric and fluorescent response to Fe3+ can be conveniently detected even by the naked eye, which provides a facile method for the visual detection of Fe3+. Job's plot, fluorescence titration and MS indicated the formation of 1:2 complexes between the probes and Fe3+. The reversibility of the reaction establishes the potential of these probes as chemosensors for Fe3+ detection. Copyright © 2013 John Wiley & Sons, Ltd.  相似文献   

8.
    
Pinak Chakrabarti 《Proteins》2015,83(4):696-710
Dystrophin is a long, rod‐shaped cytoskeleton protein implicated in muscular dystrophy (MDys). Utrophin is the closest autosomal homolog of dystrophin. Both proteins have N‐terminal actin‐binding domain (N‐ABD), a central rod domain and C‐terminal region. N‐ABD, composed of two calponin homology (CH) subdomains joined by a helical linker, harbors a few disease causing missense mutations. Although the two proteins share considerable homology (>72%) in N‐ABD, recent structural and biochemical studies have shown that there are significant differences (including stability, mode of actin‐binding) and their functions are not completely interchangeable. In this investigation, we have used extensive molecular dynamics simulations to understand the differences and the similarities of these two proteins, along with another actin‐binding protein, fimbrin. In silico mutations were performed to identify two key residues that might be responsible for the dynamical difference between the molecules. Simulation points to the inherent flexibility of the linker region, which adapts different conformations in the wild type dystrophin. Mutations T220V and G130D in dystrophin constrain the flexibility of the central helical region, while in the two known disease‐causing mutants, K18N and L54R, the helicity of the region is compromised. Phylogenetic tree and sequence analysis revealed that dystrophin and utrophin genes have probably originated from the same ancestor. The investigation would provide insight into the functional diversity of two closely related proteins and fimbrin, and contribute to our understanding of the mechanism of MDys. Proteins 2015; 83:696–710. © 2015 Wiley Periodicals, Inc.  相似文献   

9.
    
The yellow fluorescent protein phiYFPv (λemmax≃ 537 nm) with improved folding has been developed from the spectrally identical wild‐type phiYFP found in the marine jellyfish Phialidium. The latter fluorescent protein is one of only two known cases of naturally occurring proteins that exhibit emission spectra in the yellow–orange range (535–555 nm). Here, the crystal structure of phiYFPv has been determined at 2.05 Å resolution. The `yellow' chromophore formed from the sequence triad Thr65‐Tyr66‐Gly67 adopts the bicyclic structure typical of fluorophores emitting in the green spectral range. It was demonstrated that perfect antiparallel π‐stacking of chromophore Tyr66 and the proximal Tyr203, as well as Val205, facing the chromophore phenolic ring are chiefly responsible for the observed yellow emission of phiYFPv at 537 nm. Structure‐based site‐directed mutagenesis has been used to identify the key functional residues in the chromophore environment. The obtained results have been utilized to improve the properties of phiYFPv and its homologous monomeric biomarker tagYFP.  相似文献   

10.
    
One of the best‐studied examples of a class A β‐lactamase is Escherichia coli TEM‐1 β‐lactamase. In this class of enzymes, the active‐site serine residue takes on the role of a nucleophile and carries out β‐lactam hydrolysis. Here, the structures of the wild‐type and the S70G enzyme determined to 1.55 and 2.1 Å, respectively, are presented. In contrast to the previously reported 1.8 Å structure, the active site of the wild‐type enzyme (1.55 Å) structure does not contain sulfate and Ser70 appears to be in the deprotonated form. The X‐ray crystal structure of the S70G mutant has an altered Ser130 side‐chain conformation that influences the positions of water molecules in the active site. This change allows an additional water molecule to be positioned similarly to the serine hydroxyl in the wild‐type enzyme. The structure of the mutant enzyme suggests that this water molecule can assume the role of an active‐site nucleophile and carry out noncovalent catalysis. The drop in activity in the mutant enzyme is comparable to the drop observed in an analogous mutation of the nucleophilic serine in alkaline phosphatase, suggesting common chemical principles in the utilization of nucleophilic serine in the active site of different enzymes.  相似文献   

11.
    
Glucosamine‐6‐phosphate synthase (EC 2.6.1.16) catalyses the first and practically irreversible step in the hexosamine metabolism pathway, the end product of which, uridine 5′‐diphospho‐N‐acetyl d ‐glucosamine, is an essential substrate for assembly of the cell wall. The isomerase domain, consisting of residues 346–712 (42 kDa), of glucosamine‐6‐phosphate synthase from Candida albicans has been crystallized. X‐ray analysis revealed that the crystals belonged to space group I4, with unit‐cell parameters a = b = 149, c = 103 Å. Diffraction data were collected to 3.8 Å. Preliminary results from molecular replacement using the homologous bacterial monomer reveal that the asymmetric unit contains two monomers that resemble a bacterial dimer. The crystal lattice consists of pairs of such symmetry‐related dimers forming elongated tetramers.  相似文献   

12.
    
Chemokine CXCL8 and its receptor CXCR1 are key mediators in combating infection and have also been implicated in the pathophysiology of various diseases including chronic obstructive pulmonary disease (COPD) and cancer. CXCL8 exists as monomers and dimers but monomer alone binds CXCR1 with high affinity. CXCL8 function involves binding two distinct CXCR1 sites – the N‐terminal domain (Site‐I) and the extracellular/transmembrane domain (Site‐II). Therefore, higher monomer affinity could be due to stronger binding at Site‐I or Site‐II or both. We have now characterized the binding of a human CXCR1 N‐terminal domain peptide (hCXCR1Ndp) to WT CXCL8 under conditions where it exists as both monomers and dimers. We show that the WT monomer binds the CXCR1 N‐domain with much higher affinity and that binding is coupled to dimer dissociation. We also characterized the binding of two CXCL8 monomer variants and a trapped dimer to two different hCXCR1Ndp constructs, and observe that the monomer binds with ～10‐ to 100‐fold higher affinity than the dimer. Our studies also show that the binding constants of monomer and dimer to the receptor peptides, and the dimer dissociation constant, can vary significantly as a function of pH and buffer, and so the ability to observe WT monomer peaks is critically dependent on NMR experimental conditions. We conclude that the monomer is the high affinity CXCR1 agonist, that Site‐I interactions play a dominant role in determining monomer vs. dimer affinity, and that the dimer plays an indirect role in regulating monomer function.  相似文献   

13.
    
Heterodimeric proteins with homologous subunits of same fold are involved in various biological processes. The objective of this study is to understand the evolution of structural and functional features of such heterodimers. Using a non‐redundant dataset of 70 such heterodimers of known 3D structure and an independent dataset of 173 heterodimers from yeast, we note that the mean sequence identity between interacting homologous subunits is only 23–24% suggesting that, generally, highly diverged paralogues assemble to form such a heterodimer. We also note that the functional roles of interacting subunits/domains are generally quite different. This suggests that, though the interacting subunits/domains are homologous, the high evolutionary divergence characterize their high functional divergence which contributes to a gross function for the heterodimer considered as a whole. The inverse relationship between sequence identity and RMSD of interacting homologues in heterodimers is not followed. We also addressed the question of formation of homodimers of the subunits of heterodimers by generating models of fictitious homodimers on the basis of the 3D structures of the heterodimers. Interaction energies associated with these homodimers suggests that, in overwhelming majority of the cases, such homodimers are unlikely to be stable. Majority of the homologues of heterodimers of known structures form heterodimers (51.8%) and a small proportion (14.6%) form homodimers. Comparison of 3D structures of heterodimers with homologous homodimers suggests that interfacial nature of residues is not well conserved. In over 90% of the cases we note that the interacting subunits of heterodimers are co‐localized in the cell. Proteins 2015; 83:1766–1786. © 2015 Wiley Periodicals, Inc.  相似文献   

14.
    
Quantum yield is a determinant for fluorescent protein (FP) applications and enhancing FP brightness through gene engineering is still a challenge. Green2, our de novo FP synthesized by microfluidic picoarray and cloning, has a significantly lower quantum yield than enhanced green FP, though they have high homology and share the same chromophore. To increase its quantum yield, we introduced an F145Y substitution into Green2 based on rational structural analysis. Y145 significantly increased the quantum yield (0.22 vs. 0.18) and improved the photostability (t1/2, 73.0 s vs. 46.0 s), but did not affect the excitation and emission spectra. Further structural analysis showed that the F145Y substitution resulted in a significant electrical field change in the immediate environment of the chromophore. The perturbation of electrostatic charge around the chromophore lead to energy barrier changes between the ground and excited states, which resulted in the enhancement of quantum yield and photostability. Our results illustrate a typical example of engineering an FP based solely on fluorescence efficiency optimization and provide novel insights into the rational evolution of FPs.  相似文献   

15.
    
The fluorescent protein from Dendronephthya sp. (DendFP) is a member of the Kaede‐like group of photoconvertible fluorescent proteins with a His62‐Tyr63‐Gly64 chromophore‐forming sequence. Upon irradiation with UV and blue light, the fluorescence of DendFP irreversibly changes from green (506 nm) to red (578 nm). The photoconversion is accompanied by cleavage of the peptide backbone at the Cα—N bond of His62 and the formation of a terminal carboxamide group at the preceding Leu61. The resulting double Cα=Cβ bond in His62 extends the conjugation of the chromophore π system to include imidazole, providing the red fluorescence. Here, the three‐dimensional structures of native green and photoconverted red forms of DendFP determined at 1.81 and 2.14 Å resolution, respectively, are reported. This is the first structure of photoconverted red DendFP to be reported to date. The structure‐based mutagenesis of DendFP revealed an important role of positions 142 and 193: replacement of the original Ser142 and His193 caused a moderate red shift in the fluorescence and a considerable increase in the photoconversion rate. It was demonstrated that hydrogen bonding of the chromophore to the Gln116 and Ser105 cluster is crucial for variation of the photoconversion rate. The single replacement Gln116Asn disrupts the hydrogen bonding of Gln116 to the chromophore, resulting in a 30‐fold decrease in the photoconversion rate, which was partially restored by a further Ser105Asn replacement.  相似文献   

16.
    
Monomeric Azami‐Green (mAG) from the stony coral Galaxea fascicularis is the first known monomeric green‐emitting fluorescent protein that is not a variant of Aequorea victoria green fluorescent protein (avGFP). These two green fluorescent proteins are only 27% identical in their amino‐acid sequences. mAG is more similar in its amino‐acid sequence to four fluorescent proteins: Dendra2 (a green‐to‐red irreversibly photoconverting fluorescent protein), Dronpa (a bright‐and‐dark reversibly photoswitchable fluorescent protein), KikG (a tetrameric green‐emitting fluorescent protein) and Kaede (another green‐to‐red irreversibly photoconverting fluorescent protein). To reveal the structural basis of stable green emission by mAG, the 2.2 Å crystal structure of mAG has been determined and compared with the crystal structures of avGFP, Dronpa, Dendra2, Kaede and KikG. The structural comparison revealed that the chromophore formed by Gln62‐Tyr63‐Gly64 (QYG) and the fixing of the conformation of the imidazole ring of His193 by hydrogen bonds and van der Waals contacts involving His193, Arg66 and Thr69 are likely to be required for the stable green emission of mAG. The crystal structure of mAG will contribute to the design and development of new monomeric fluorescent proteins with faster maturation, brighter fluorescence, improved photostability, new colours and other preferable properties as alternatives to avGFP and its variants.  相似文献   

17.
    
The high‐potential iron–sulfur protein (HiPIP) is an electron carrier between the photosynthetic reaction centre and the cytochrome bc1 complex in the electron‐transfer chain of photosynthesis. The purified HiPIP from Thermochromatium tepidum (formerly Chromatium tepidum) was crystallized in a solution of 1.4 M ammonium sulfate and 0.1 M sodium citrate pH 3.5. The crystals diffract X‐rays beyond 1.4 Å resolution and belong to the orthorhombic space group P212121, with unit‐cell parameters a = 47.12 (6), b = 59.59 (10), c = 23.62 (3) Å. The structure was preliminarily solved by the molecular‐replacement method. The crystal structure of HiPIP from T. tepidum showed that the proteins exist as monomers, although HiPIPs from several other species can form dimers.  相似文献   

18.
    
Peptidyl‐prolyl cis‐trans isomerases (PPIases) are the enzymes that increase the rate of isomerization of the peptide bond N‐terminal to the proline substrate. Par14 and its isoform Par17 belong to the Parvulin family of PPIases. Par14 can bind AT‐rich double‐stranded DNA and was shown to be part of the pre‐ribosomal ribonucleoprotein (pre‐rRNP) complexes, where it functions as an RNA processing factor that is involved in ribosome biogenesis. Its longer isoform Par17 is expressed only in cells of hominids, where it is targeted to the mitochondria. To find binding partners (peptides or proteins) for Par17, we applied the phage display technology. We panned 7‐mer and 12‐mer peptide libraries against Par17. The consensus sequence XHSXVHØ, where X can be any amino acid and Ø is a hydrophobic amino acid, was enriched from both libraries. We demonstrate the binding of this motif to the PPIase domain of Par17 using phage ELISA and NMR spectroscopy. We propose that residues Met90, Val91, Phe94, Gln95, Glu96, and Ala98 of Par17 are involved in substrate recognition, and that the phage display‐selected motif XHSXVHØ can be recognized by Par17 PPIase domain in vivo. Copyright © 2013 European Peptide Society and John Wiley & Sons, Ltd.  相似文献   

19.
    
Crystals of the molybdo‐/iron–sulfur protein pyrogallol:phloroglucinol hydroxyltransferase (transhydroxylase; EC 1.97.1.2) from Pelobacter acidigallici were grown by vapour diffusion in an N2/H2 atmosphere using polyethylene glycol as a precipitant. In this microorganism, transhydroxylase converts pyrogallol to phloroglucinol in a unique reaction without oxygen transfer from water. Growth of crystals suitable for X‐ray analysis was strongly dependent on the presence of dithionite as a reducing agent. The crystals belonged to space group P1 and MAD data were collected on the iron K edge to resolutions higher than 2.5 Å.  相似文献   

20.
    
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