scholarly journals Site-Directed Mutagenesis of Myoglobin for Studies of Their Interaction with Iron(III) by Multi-Spectroscopic Techniques

2013 ◽  
Vol 2013 ◽  
pp. 1-11
Author(s):  
Qian Tang ◽  
Xiao-Jun Peng ◽  
Hong-Yu Cao ◽  
Yan-Jie Yang ◽  
Jing Ma ◽  
...  
Keyword(s):  
Amino Acids ◽  
Metal Ion ◽  
Binding Capacity ◽  
Three Dimensional ◽  
Site Directed Mutagenesis ◽  
Spectroscopic Techniques ◽  
Wild Type ◽  
Bimolecular Quenching ◽  
And Function ◽  
Binding Distance

In order to investigate how the amino acids on the surface of myoglobin molecule influence myoglobin's structure and function, a variety of spectroscopy techniques were applied in the study of the interaction between Fe(III) and myoglobin (wild type and its mutants, D44K, D60K, and K56D). The results demonstrate that Fe(III) can quench the fluorescence of wild type and mutants of myoglobin, and the quenching mechanisms are static quenching. It is found that the binding distance between Fe(III) and myoglobin mutants gets smaller, the binding capacity increases by the values of binding constant and the bimolecular quenching constant as well as the binding distance. Those data also indicate that the metal ion Fe(III) can interact strongly with myoglobin mutants. The three-dimensional conformation change after surface amino acids are replaced is detected by the UV absorption spectroscopy and fluorescence spectroscopy, which make mutants become more dynamic and change its function and interaction with Fe(III) strongly.

Characterization of active-site residues in diadenosine tetraphosphate hydrolase from Lupinus angustifolius

2001 ◽  
Vol 357 (2) ◽  
pp. 399-405 ◽  
Author(s):  
Danuta MAKSEL ◽  
Paul R. GOOLEY ◽  
James D. SWARBRICK ◽  
Andrzej GURANOWSKI ◽  
Christine GANGE ◽  
...  
Keyword(s):  
Amino Acids ◽  
Metal Ion ◽  
Solution Structure ◽  
Ph Dependence ◽  
Three Dimensional ◽  
Lupinus Angustifolius ◽  
Site Directed Mutagenesis ◽  
Nudix Hydrolase ◽  
Active Site Residues ◽  
Dimensional Solution

Site-directed mutagenesis has been used to characterize the functions of key amino acid residues in the catalytic site of the ‘nudix’ hydrolase, (asymmetrical) diadenosine 5′,5‴-P1,P4-tetraphosphate (Ap4A) hydrolase (EC 3.6.1.17) from Lupinus angustifolius, the three-dimensional solution structure of which has recently been solved. Residues within the nudix motif, Gly-(Xaa)5-Glu-(Xaa)7-Arg-Glu-Uaa-Xaa-(Glu)2-Xaa-Gly (where Xaa represents unspecified amino acids and Uaa represents the bulky aliphatic amino acids Ile, Leu or Val) conserved in ‘nudix enzymes’, and residues important for catalysis from elsewhere in the molecule, were mutated and the expressed proteins characterized. The results reveal a high degree of functional conservation between lupin asymmetric Ap4A hydrolase and the 8-oxo-dGTP hydrolase from Escherichia coli. Charged residues in positions equivalent to those that ligate an enzyme-bound metal ion in the E. coli 8-oxo-dGTP hydrolase [Harris, Wu, Massiah and Mildvan (2000) Biochemistry 39, 1655–1674] were shown to contribute to catalysis to similar extents in the lupin enzyme. Mutations E55Q, E59Q and E125Q all reduced kcat markedly, whereas mutations R54Q, E58Q and E122Q had smaller effects. None of the mutations produced a substantial change in the Km for Ap4A, but several extensively modified the pH-dependence and fluoride-sensitivities of the hydrolase. It was concluded that the precisely positioned glutamate residues Glu-55, Glu-59 and Glu-125 are conserved as functionally significant components of the hydrolytic mechanism in both of these members of the nudix family of hydrolases.

scholarly journals Isolation and Characterization of Mutations in theEscherichia coli Regulatory Protein XapR

1999 ◽  
Vol 181 (14) ◽  
pp. 4397-4403 ◽  
Author(s):  
Casper Jørgensen ◽  
Gert Dandanell
Keyword(s):  
Amino Acids ◽  
Purine Nucleoside Phosphorylase ◽  
Mutational Analysis ◽  
Regulatory Protein ◽  
Three Dimensional ◽  
Dimensional Structure ◽  
Wild Type ◽  
Isolation And Characterization ◽  
In The Wild

ABSTRACT In this work, the LysR-type protein XapR has been subjected to a mutational analysis. XapR regulates the expression of xanthosine phosphorylase (XapA), a purine nucleoside phosphorylase inEscherichia coli. In the wild type, full expression of XapA requires both a functional XapR protein and the inducer xanthosine. Here we show that deoxyinosine can also function as an inducer in the wild type, although not to the same extent as xanthosine. We have isolated and characterized in detail the mutants that can be induced by other nucleosides as well as xanthosine. Sequencing of the mutants has revealed that two regions in XapR are important for correct interactions between the inducer and XapR. One region is defined by amino acids 104 and 132, and the other region, containing most of the isolated mutations, is found between amino acids 203 and 210. These regions, when modelled into the three-dimensional structure of CysB from Klebsiella aerogenes, are placed close together and are most probably directly involved in binding the inducer xanthosine.

scholarly journals Structural identification of the myo-inositol 1,4,5-trisphosphate-binding domain in rat brain inositol 1,4,5-trisphosphate 3-kinase

1997 ◽  
Vol 326 (1) ◽  
pp. 221-225 ◽  
Author(s):  
Shinji TOGASHI ◽  
Kazunaga TAKAZAWA ◽  
Toyoshi ENDO ◽  
Christophe ERNEUX ◽  
Toshimasa ONAYA
Keyword(s):  
Amino Acids ◽  
Rat Brain ◽  
Kinase Activity ◽  
Catalytic Domain ◽  
Site Directed Mutagenesis ◽  
Wild Type ◽  
Wild Type Enzyme ◽  
Apparent Homogeneity ◽  
Inositol 1,4,5 Trisphosphate ◽  
Charged Amino Acid Residue

A series of key amino acids involved in Ins(1,4,5)P3 (InsP3) binding and catalytic activity of rat brain InsP3 3-kinase has been identified. The catalytic domain is at the C-terminal end and restricted to a maximum of 275 amino acids [Takazawa and Erneux (1991) Biochem. J. 280, 125–129]. In this study, newly prepared 5′-deletion and site-directed mutants have been compared both for InsP3 binding and InsP3 3-kinase activity. When the protein was expressed from L259 to R459, the activity was lost but InsP3 binding was conserved. Another deletion mutant that had lost only four amino acids after L259 had lost InsP3 binding, and this finding suggests that these residues (i.e. L259DCK262) are involved in InsP3 binding. To further support the data, we have produced two mutants by site-directed mutagenesis on residues C261 and K262. The two new enzymes were designated M4 (C261S) and M5 (K262A). M4 showed similar Vmax and Km values for InsP3 and ATP to wild-type enzyme. In contrast, M5 was totally inactive but had kept the ability to bind to calmodulin–Sepharose. C-terminal deletion mutants that had lost five, seven or nine amino acids showed a large decrease in InsP3 binding and InsP3 3-kinase activity. One mutant that had lost five amino acids (M2) was purified to apparent homogeneity: Km values for both substrates appeared unchanged but Vmax was decreased approx. 40-fold compared with the wild-type enzyme. The results indicate that (1) a positively charged amino acid residue K262 is essential for InsP3 binding and (2) amino acids at the C-terminal end of the protein are necessary to act as a catalyst in the InsP3 3-kinase reaction.

scholarly journals C-terminal region regulates the functional expression of human noradrenaline transporter splice variants

2006 ◽  
Vol 401 (1) ◽  
pp. 185-195 ◽  
Author(s):  
Chiharu Sogawa ◽  
Kei Kumagai ◽  
Norio Sogawa ◽  
Katsuya Morita ◽  
Toshihiro Dohi ◽  
...  
Keyword(s):  
Amino Acids ◽  
Cell Surface ◽  
Splice Variants ◽  
Functional Expression ◽  
Surface Expression ◽  
Terminal Region ◽  
Site Directed Mutagenesis ◽  
Cell Surface Expression ◽  
C Terminus ◽  
And Function

The NET [noradrenaline (norepinephrine) transporter], an Na+/Cl−-dependent neurotransmitter transporter, has several isoforms produced by alternative splicing in the C-terminal region, each differing in expression and function. We characterized the two major isoforms of human NET, hNET1, which has seven C-terminal amino acids encoded by exon 15, and hNET2, which has 18 amino acids encoded by exon 16, by site-directed mutagenesis in combination with NE (noradrenaline) uptake assays and cell surface biotinylation. Mutants lacking one third or more of the 24 amino acids encoded by exon 14 exhibited neither cell surface expression nor NE uptake activity, with the exception of the mutant lacking the last eight amino acids of hNET2, whose expression and uptake resembled that of the WT (wild-type). A triple alanine replacement of a candidate motif (ENE) in this region mimicked the influences of the truncation. Deletion of either the last three or another four amino acids of the C-terminus encoded by exon 15 in hNET1 reduced the cell surface expression and NE uptake, whereas deletion of all seven residues reduced the transport activity but did not affect the cell surface expression. Replacement of RRR, an endoplasmic reticulum retention motif, by alanine residues in the C-terminus of hNET2 resulted in a similar expression and function compared with the WT, while partly recovering the effects of the mutation of ENE. These findings suggest that in addition to the function of the C-terminus, the common proximal region encoded by exon 14 regulates the functional expression of splice variants, such as hNET1 and hNET2.

scholarly journals Site-directed mutagenesis of proposed active-site residues of penicillin-binding protein 5 from Escherichia coli

1994 ◽  
Vol 303 (2) ◽  
pp. 357-362 ◽  
Author(s):  
M P G van der Linden ◽  
L de Haan ◽  
O Dideberg ◽  
W Keck
Keyword(s):  
Active Site ◽  
Catalytic Mechanism ◽  
Binding Capacity ◽  
Binding Protein ◽  
Complete Loss ◽  
Site Directed Mutagenesis ◽  
Directed Mutagenesis ◽  
Penicillin Binding Protein ◽  
Wild Type ◽  
Active Site Residues

Alignment of the amino acid sequence of penicillin-binding protein 5 (PBP5) with the sequences of other members of the family of active-site-serine penicillin-interacting enzymes predicted the residues playing a role in the catalytic mechanism of PBP5. Apart from the active-site (Ser44), Lys47, Ser110-Gly-Asn, Asp175 and Lys213-Thr-Gly were identified as the residues making up the conserved boxes of this protein family. To determine the role of these residues, they were replaced using site-directed mutagenesis. The mutant proteins were assayed for their penicillin-binding capacity and DD-carboxypeptidase activity. The Ser44Cys and the Ser44Gly mutants showed a complete loss of both penicillin-binding capacity and DD-carboxypeptidase activity. The Lys47Arg mutant also lost its DD-carboxypeptidase activity but was able to bind and hydrolyse penicillin, albeit at a considerably reduced rate. Mutants in the Ser110-Gly-Asn fingerprint were affected in both acylation and deacylation upon reaction with penicillin and lost their DD-carboxypeptidase activity with the exception of Asn112Ser and Asn112Thr. The Asp175Asn mutant showed wild-type penicillin-binding but a complete loss of DD-carboxypeptidase activity. Mutants of Lys213 lost both penicillin-binding and DD-carboxypeptidase activity except for Lys213His, which still bound penicillin with a k+2/K' of 0.2% of the wild-type value. Mutation of His216 and Thr217 also had a strong effect on DD-carboxypeptidase activity. Thr217Ser and Thr217Ala showed augmented hydrolysis rates for the penicillin acyl-enzyme. This study reveals the residues in the conserved fingerprints to be very important for both DD-carboxypeptidase activity and penicillin-binding, and confirms them to play crucial roles in catalysis.

scholarly journals Mapping leptin-interacting sites in recombinant leptin-binding domain (LBD) subcloned from chicken leptin receptor

2005 ◽  
Vol 390 (2) ◽  
pp. 475-484 ◽  
Author(s):  
L. Niv-Spector ◽  
N. Raver ◽  
M. Friedman-Einat ◽  
J. Grosclaude ◽  
E. E. Gussakovsky ◽  
...  
Keyword(s):  
Amino Acids ◽  
Surface Plasmon Resonance ◽  
Surface Plasmon ◽  
Plasmon Resonance ◽  
Leptin Receptor ◽  
Binding Capacity ◽  
Gel Filtration ◽  
Binding Domain ◽  
Site Directed Mutagenesis ◽  
Surface Plasmon Resonance Analysis

The binding domain of the chicken leptin receptor [chLBD (chicken leptin-binding domain)], subcloned from the full-size chicken leptin receptor and prepared in an Escherichia coli system, was subjected to site-directed mutagenesis to identify the amino acids involved in leptin binding. A total of 22 electrophoretically pure, >90% monomer-containing mutants were expressed, refolded and purified. The effects of the mutations were tested by the ability to form complexes with ovine leptin, and the kinetic parameters of interaction were determined by surface plasmon resonance. Six mutants were used to determine whether mutations of several amino acids that differ between chLBD and mammalian LBDs will affect affinity: none showed any such effect, except the mutant A105D (Ala105→Asp), which exhibited some decrease in affinity. Surface plasmon resonance analysis identified six mutants in which binding activity was totally abolished (F73A, Y14A/F73A, V76A/F77A, L78A/L79A, V76A/F77A/L78A/L79A and A105D/D106V) and six mutants (Y14A, R41A, R41A/S42A/K43A, V103A, V135A/F136A and F136A) in which affinity for the hormone was reduced, mainly by increased dissociation rates. Gel-filtration experiments indicated the formation of a 1:1 ovine or human leptin–chLBD complex with a molecular mass of approx. 41 kDa. Gel-filtration experiments yielded 1:1 complexes with those mutants in which affinity had decreased, but not with the six mutants, which had totally lost their binding capacity. Modelling the leptin–chLBD complex indicated that the binding domain of the latter is located mainly in the L3 loop, which contributes nine amino acid residues interacting with leptin. Contact-surface analysis identified the residues having the highest contribution to the recognition site to be Phe73, Phe77 and Leu79.

Site-directed mutagenesis of farnesyl diphosphate synthase; effect of substitution on the three carboxyl-terminal amino acids

1994 ◽  
Vol 72 (1) ◽  
pp. 75-79 ◽  
Author(s):  
Tanetoshi Koyama ◽  
Kazuhiro Saito ◽  
Kyozo Ogura ◽  
Shusei Obata ◽  
Ayumi Takeshita
Keyword(s):  
Amino Acids ◽  
Bacillus Stearothermophilus ◽  
Farnesyl Diphosphate ◽  
Site Directed Mutagenesis ◽  
Farnesyl Diphosphate Synthase ◽  
Wild Type ◽  
Wild Type Enzyme ◽  
Directed Mutation ◽  
Catalytic Function ◽  
Terminal Amino

Site-directed mutation was introduced into the gene for the farnesyl diphosphate synthase of Bacillus stearothermophilus. To investigate the significance of the three C-terminal amino acids, where arginine is completely conserved throughout the farnesyl diphosphate synthases of prokaryotes and eukaryotes, three kinds of mutant enzymes, R295V, D296G, and H297L, which have replacements of arginine-295 with valine, aspartate-296 with glycine, and histidine-297 with leucine, respectively, were overproduced and purified to homogeneity. All of the three mutant enzymes showed similar catalytic activities to that of the wild-type enzyme, indicating that the basic amino acids including the conserved arginine in the C-terminal region are not essential for catalytic function. They were also similar to the wild-type enzyme with respect to pH optima, thermostability, reaction product, and kinetic parameters for allylic substrates. However, their Km values for isopentenyl diphosphate are approximately twice that of the wild type.

scholarly journals Analisis In-Silico Struktur Tiga Dimensi Reseptor Trk A dan Trk B Protein Neurotrophin 3 Pada Gallus gallus (Chicken)

2020 ◽  
Vol 12 (2) ◽  
pp. 78-84
Author(s):  
Muhammad F. Rahman ◽  
Amiruddin Kasim ◽  
Muchlis L. Djirimu ◽  
I. Made Budiarsa
Keyword(s):  
Amino Acids ◽  
In Silico ◽  
Three Dimensional ◽  
Gallus Gallus ◽  
Dimensional Structure ◽  
Neurotrophin 3 ◽  
Trk A ◽  
And Function ◽  
Trk B ◽  
Ucsf Chimera

NT3 protein is expressed by Neurotrophin 3 (NTF-3) which plays a role in the process of differentiation, survival of peripheral and neuropathological of neurons. The information of structure and function of NT-3 proteins is still very limited, especially in Gallus gallus. This study aims to predict the three-dimensional structure of the Trk A and Trk B proteins in Gallus gallus. The target protein obtained from the UniProt server with access codes Q91009 (Trk A) and Q91987 (Trk B) using the 6kzc 1.A (PDB ID) template was analyzed in silico through a homology approach and describing the structural assessment using Chimera UCSF software. The analysis showed that the Trk A protein had a QMEAN value of -0.08, composed of 778 amino acids, mass 87334.30 Daltons, and Seq Identity 79.93%. Trk B had a QMEAN value of 0.16, consisting of 818 amino acids, mass 91732.05 Daltons, and Seq Identity 84.30%. Key words: NT3; homology; UCSF chimera; G. gallus

Phosphorylation of FANCA on S1449 Is Important for Fanconi Anemia (FA) Pathway Function.

Blood ◽  
2006 ◽  
Vol 108 (11) ◽  
pp. 186-186
Author(s):  
Natalie B. Collins ◽  
Andrei Tomashevski ◽  
Gary M. Kupfer
Keyword(s):  
Dna Damage ◽  
Homologous Recombination ◽  
Dna Damage Response ◽  
Fanconi Anemia ◽  
Phosphorylation Site ◽  
Site Directed Mutagenesis ◽  
Wild Type ◽  
Damage Response ◽  
And Function ◽  
Mutant Cells

Abstract Previous work in our lab and others has shown that the Fanconi anemia proteins, FANCG and FANCA, are phosphoproteins. FANCG is phosphorylated at mitosis, and these phosphorylations are required for proper exit from chromatin at mitosis. FANCG is also phosphorylated after DNA damage, with the phosphorylation site required for wild-type sensitivity to DNA damaging agents. FANCA is also phosphorylated after DNA damage and localized to chromatin, but the site and significance of this phosphorylation were previously unknown. Mass spectrometry of FANCA revealed one phosphopeptide with phosphorylation on serine 1449. Site-directed mutagenesis of this residue to alanine (S1449A) abolished a slower mobility form of FANCA seen after MMC treatment. Furthermore, the S1449A mutant failed to completely correct the MMC hypersensitivity of FA-A mutant cells. S1449A mutant cells displayed lower than wild-type levels of FANCD2 monoubiquitination following DNA damage, and an increased number of gross chromosomal aberrations were seen in metaphase spreads from S1449A mutant cells when compared to wild type cells. Using a GFP reporter substrate to measure homologous recombination, cells expressing the S1449A FANCA failed to completely correct the homologous recombination defect seen in FA cells. Taken together, cells expressing FANCA S1449A display a variety of FA-associated phenotypes, suggesting that the phosphorylation of S1449 is a functionally significant event. The DNA damage response in human cells is, in large part, coordinated by phosphorylation events initiated by apical kinases ATM and ATR. S1449 is found in a consensus ATM site, therefore studies are underway to determine if ATM or ATR is the kinase responsible for FANCA phosphorylation at S1449. Phosphorylation is a crucial process in transducing the DNA damage response, and phosphorylation of FA proteins appears critical to both localization and function of the proteins. Understanding how phosphorylation marks are placed on FANCA will give insight into the role of FANCA in the DNA damage response.

scholarly journals Basic Residues of the Helix Six Domain of Influenza Virus M1 Involved in Nuclear Translocation of M1 Can Be Replaced by PTAP and YPDL Late Assembly Domain Motifs

2003 ◽  
Vol 77 (12) ◽  
pp. 7078-7092 ◽  
Author(s):  
Eric Ka-Wai Hui ◽  
Subrata Barman ◽  
Tae Yong Yang ◽  
Debi P. Nayak
Keyword(s):  
Amino Acids ◽  
Influenza Virus ◽  
Reverse Genetics ◽  
Infectious Virus ◽  
Nuclear Translocation ◽  
Critical Role ◽  
Site Directed Mutagenesis ◽  
Plaque Morphology ◽  
Wild Type ◽  
Wild Type Phenotype

ABSTRACT Influenza type A virus matrix (M1) protein possesses multiple functional motifs in the helix 6 (H6) domain (amino acids 91 to 105), including nuclear localization signal (NLS) (101-RKLKR-105) involved in translocating M1 from the cytoplasm into the nucleus. To determine the role of the NLS motif in the influenza virus life cycle, we mutated these and the neighboring sequences by site-directed mutagenesis, and influenza virus mutants were generated by reverse genetics. Our results show that infectious viruses were rescued by reverse genetics from all single alanine mutations of amino acids in the H6 domain and the neighboring region except in three positions (K104A and R105A within the NLS motif and E106A in loop 6 outside the NLS motif). Among the rescued mutant viruses, R101A and R105K exhibited reduced growth and small-plaque morphology, and all other mutant viruses showed the wild-type phenotype. On the other hand, three single mutations (K104A, K105A, and E106A) and three double mutations (R101A/K102A, K104A/K105A, and K102A/R105A) failed to generate infectious virus. Deletion (ΔYRKL) or mutation (4A) of YRKL also abolished generation of infectious virus. However, replacement of the YRKL motif with PTAP or YPDL as well as insertion of PTAP after 4A mutation yielded infectious viruses with the wild-type phenotype. Furthermore, mutant M1 proteins (R101A/K102A, ΔYRKL, 4A, PTAP, 4A+PTAP, and YPDL) when expressed alone from cloned cDNAs were only cytoplasmic, whereas the wild-type M1 expressed alone was both nuclear and cytoplasmic as expected. These results show that the nuclear translocation function provided by the positively charged residues within the NLS motif does not play a critical role in influenza virus replication. Furthermore, these sequences of H6 domain can be replaced by late (L) domain motifs and therefore may provide a function similar to that of the L domains of other negative-strand RNA and retroviruses.

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