scholarly journals An Activated Glutamate Residue Identified in Photosystem II at the Interface between the Manganese-stabilizing Subunit and the D2 Polypeptide

2007 ◽  
Vol 282 (38) ◽  
pp. 27802-27809 ◽  
Author(s):  
Sascha Rexroth ◽  
Catherine C. L. Wong ◽  
Jessica H. Park ◽  
John R. Yates ◽  
Bridgette A. Barry
Keyword(s):  
Amino Acid ◽  
Photosystem Ii ◽  
Oxygenic Photosynthesis ◽  
Photosynthetic Oxygen Evolution ◽  
Amino Acid Residues ◽  
Post Translational Modification ◽  
Steady State Rate ◽  
Reactive Groups ◽  
Psii Subunits

Photosystem II (PSII) catalyzes the oxidation of water during oxygenic photosynthesis. PSII is composed both of intrinsic subunits, such as D1, D2, and CP47, and extrinsic subunits, such as the manganese-stabilizing subunit (MSP). Previous work has shown that amines covalently bind to amino acid residues in the CP47, D1, and D2 subunits of plant and cyanobacterial PSII, and that these covalent reactions are prevented by the addition of chloride in plant preparations depleted of the 18- and 24-kDa extrinsic subunits. It has been proposed that these reactive groups are carbonyl-containing, post-translationally modified amino acid side chains (Ouellette, A. J. A., Anderson, L. B., and Barry, B. A. (1998) Proc. Natl. Acad. Sci. U. S. A. 95, 2204–2209 and Anderson, L. B., Ouellette, A. J. A., and Barry, B. A. (2000) J. Biol. Chem. 275, 4920–4927). To identify the amino acid binding site in the spinach D2 subunit, we have employed a biotin-amine labeling reagent, which can be used in conjunction with avidin affinity chromatography to purify biotinylated peptides from the PSII complex. Multidimensional chromato-graphic separation and multistage mass spectrometry localizes a novel post-translational modification in the D2 subunit to glutamate 303. We propose that this glutamate is activated for amine reaction by post-translational modification. Because the modified glutamate is located at a contact site between the D2 and manganese-stabilizing subunits, we suggest that the modification is important in vivo in stabilizing the interaction between these two PSII subunits. Consistent with this conclusion, mutations at the modified glutamate alter the steady-state rate of photosynthetic oxygen evolution.

Structure - function analysis of photosystem II subunit S (PsbS) in vivo

2002 ◽  
Vol 29 (10) ◽  
pp. 1131 ◽  
Author(s):  
Xiao-Ping Li ◽  
Alba Phippard ◽  
Jae Pasari ◽  
Krishna K. Niyogi
Keyword(s):  
Amino Acid ◽  
Photosystem Ii ◽  
Function Analysis ◽  
Amino Acid Sequences ◽  
Forward Genetics ◽  
Photochemical Quenching ◽  
Amino Acid Residues ◽  
The Stability ◽  
Psbs Gene

In land plants, photosystem II subunit S (PsbS) plays a key role in xanthophyll- and pH-dependent non-photochemical quenching (qE) of excess absorbed light energy. Arabidopsis thaliana (L.) Heynh. npq4 mutants are defective in the psbS gene and have impaired qE. Exactly how the PsbS protein is involved in qE is unclear, but it has been proposed that PsbS binds H+ and/or de-epoxidized xanthophylls in excess light as part of the qE mechanism. To identify amino acid residues that are important for PsbS function, we sequenced the psbS gene from eight npq4 point mutant alleles isolated by forward genetics screening, including two new alleles. In the four transmembrane helices of PsbS, several amino acid residues were found to affect the stability and/or function of the protein. By comparing the predicted amino acid sequences of PsbS from several plant species and studying the proposed topological structure of PsbS, eight possible H+-binding amino acid residues on the lumenal side of the protein were identified and then altered by site-directed mutagenesis in vitro. The mutant psbS genes were transformed into npq4-1, a psbS deletion mutant, to test the stability and function of the mutant PsbS proteins in�vivo. The results demonstrate that two conserved, protonatable amino acids, E122 and E226, are especially critical for the function of PsbS.

Oxytocin analogues with non-coded amino acid residues in position 8: [8-Neopentylglycine]oxytocin and [8-cycloleucine]oxytocin

1987 ◽  
Vol 52 (9) ◽  
pp. 2317-2325 ◽  
Author(s):  
Jan Hlaváček ◽  
Jan Pospíšek ◽  
Jiřina Slaninová ◽  
Walter Y. Chan ◽  
Victor J. Hruby
Keyword(s):  
Amino Acid ◽  
Solid Phase Synthesis ◽  
Solid Phase ◽  
The Other ◽  
Amino Acid Residues ◽  
Phase Synthesis ◽  
Other Hand ◽  
Fragment Condensation

[8-Neopentylglycine]oxytocin (II) and [8-cycloleucine]oxytocin (III) were prepared by a combination of solid-phase synthesis and fragment condensation. Both analogues exhibited decreased uterotonic potency in vitro, each being about 15-30% that of oxytocin. Analogue II also displayed similarly decreased uterotonic potency in vivo and galactogogic potency. On the other hand, analogue III exhibited almost the same potency as oxytocin in the uterotonic assay in vivo and in the galactogogic assay.

Synthesis of Fragments of the Vasoactive Intestinal Peptide (VIP) and Analysis of Their Residual Biological Activities

1995 ◽  
Vol 60 (7) ◽  
pp. 1229-1235 ◽  
Author(s):  
Ivana Zoulíková ◽  
Ivan Svoboda ◽  
Jiří Velek ◽  
Václav Kašička ◽  
Jiřina Slaninová ◽  
...  
Keyword(s):  
Amino Acid ◽  
Biological Activities ◽  
Residual Activity ◽  
Detailed Examination ◽  
Amino Acid Residues ◽  
Linear Peptide ◽  
Zone Electrophoresis ◽  
Capillary Zone

The vasoactive intestinal (poly)peptide (VIP) is a linear peptide containing 28 amino acid residues, whose primary structure indicates a low metabolic stability. The following VIP fragments, as potential metabolites, and their analogues were prepared by synthesis on a solid: [His(Dnp)1]VIP(1-10), VIP(11-14), [D-Arg12]VIP(11-14), [Lys(Pac)15,21,Arg20]VIP(15-22), and VIP(23-28). After purification, the peptides were characterized by amino acid analysis, mass spectrometry, RP HPLC, and capillary zone electrophoresis. In some tests, detailed examination of the biological activity of the substances in vivo and in vitro gave evidence of a low, residual activity of some fragments, viz. a depressoric activity in vivo for [His(Dnp)1]VIP(1-10) and a stimulating activity for the release of α-amylase in vitro and in vivo for [Lys(Pac)15,21,Arg20]VIP(15-22) and VIP(23-28).

scholarly journals Design and Synthesis of Pyrazole-3-one Derivatives as Hypoglycaemic Agents

2015 ◽  
Vol 2015 ◽  
pp. 1-10 ◽  
Author(s):  
Prasanna A. Datar ◽  
Sonali R. Jadhav
Keyword(s):  
Amino Acid ◽  
Docking Studies ◽  
Diabetic Rats ◽  
Hypoglycemic Activity ◽  
Amino Acid Residues ◽  
Docking Score ◽  
Design And Synthesis ◽  
Hypoglycaemic Agents ◽  
Standard Compound

Pyrazole-3-one compounds were designed on the basis of docking studies of previously reported antidiabetic pyrazole compounds. The amino acid residues found during docking studies were used as guidelines for the modification of aromatic substitutions on pyrazole-3-one structure. Depending on the docking score, the designed compounds were selectively prioritized for synthesis. The synthesized compounds were subjected to in vivo hypoglycemic activity using alloxan induced diabetic rats and metformin as a standard. Compound 4 having sulphonamide derivative was found to be the most potent compound among the series.

scholarly journals In VivoEffects of Leptin-Related Synthetic Peptides on Body Weight and Food Intake in Female ob/obMice: Localization of Leptin Activity to Domains Between Amino Acid Residues 106–140*

Endocrinology ◽  
1997 ◽  
Vol 138 (4) ◽  
pp. 1413-1418 ◽  
Author(s):  
Patricia Grasso ◽  
Matthew C. Leinung ◽  
Stacy P. Ingher ◽  
Daniel W. Lee
Keyword(s):  
Body Weight ◽  
Weight Gain ◽  
Amino Acid ◽  
Food Intake ◽  
Stop Codon ◽  
Premature Stop Codon ◽  
Amino Acid Residues ◽  
Inactive Form ◽  
In Vivo Effects

Abstract In C57BL/6J ob/ob mice, a single base mutation of the ob gene in codon 105 results in the replacement of arginine by a premature stop codon and production of a truncated inactive form of leptin. These observations suggest that leptin activity may be localized, at least in part, to domains distal to amino acid residue 104. To investigate this possibility, we synthesized six overlapping peptide amides corresponding to residues 106–167 of leptin, and examined their effects on body weight and food intake in female C57BL/6J ob/ob mice. When compared with vehicle-injected control mice, weight gain by mice receiving 28 daily 1-mg ip injections of LEP-(106–120), LEP-(116–130), or LEP-(126–140) was significantly (P < 0.01) reduced with no apparent toxicity. Weight gain by mice receiving LEP-(136–150), LEP-(146–160), or LEP-(156–167) was not significantly different from that of vehicle-injected control mice. The effects of LEP-(106–120), LEP-(116–130), or LEP-(126–140) were most pronounced during the first week of peptide treatment. Within 7 days, mice receiving these peptides lost 12.3%, 13.8%, and 9.8%, respectively, of their initial body weights. After 28 days, mice given vehicle alone, LEP-(136–150), LEP-(146–160), or LEP-(156–167) were 14.7%, 20.3%, 25.0%, and 24.8% heavier, respectively, than they were at the beginning of the study. Mice given LEP-(106–120) or LEP-(126–140) were only 1.8% and 4.2% heavier, respectively, whereas mice given LEP-(116–130) were 3.4% lighter. Food intake by mice receiving LEP-(106–120), LEP-(116–130), or LEP-(126–140), but not by mice receiving LEP-(136–150), LEP-(146–160), or LEP-(156–167), was reduced by 15%. The results of this study indicate 1) that leptin activity is localized, at least in part, in domains between residues 106–140; 2) that leptin-related peptides have in vivo effects similar to those of native leptin; and 3) offer hope for development of peptide analogs of leptin having potential application in human or veterinary medicine.

Structural basis of cofactor-mediated stabilization and substrate recognition of the α-tubulin acetyltransferase αTAT1

2015 ◽  
Vol 467 (1) ◽  
pp. 103-113 ◽  
Author(s):  
Satoru Yuzawa ◽  
Sachiko Kamakura ◽  
Junya Hayase ◽  
Hideki Sumimoto
Keyword(s):  
Catalytic Domain ◽  
Substrate Recognition ◽  
Structural Basis ◽  
Stable Complex ◽  
Amino Acid Residues ◽  
Substrate Selection ◽  
Post Translational Modification ◽  
Acetyl Group

The functions of microtubules are controlled in part by tubulin post-translational modification including acetylation of Lys40 in α-tubulin. αTAT1 (α-tubulin acetyltransferase 1), an enzyme evolutionarily conserved among eukaryotes, has recently been identified as the major α-tubulin Lys40 acetyltransferase, in which AcCoA (acetyl-CoA) serves as an acetyl group donor. The regulation and substrate recognition of this enzyme, however, have not been fully understood. In the present study, we show that AcCoA and CoA each form a stable complex with human αTAT1 to maintain the protein integrity both in vivo and in vitro. The invariant residues Arg132 and Ser160 in αTAT1 participate in the stable interaction not only with AcCoA but also with CoA, which is supported by analysis of the present crystal structures of the αTAT1 catalytic domain in complex with CoA. Alanine substitution for Arg132 or Ser160 leads to a drastic misfolding of the isolated αTAT1 catalytic domain in the absence of CoA and AcCoA but not in the presence of excess amounts of either cofactor. A mutant αTAT1 carrying the R132A or S160A substitution is degraded much faster than the wild-type protein when expressed in mammalian Madin–Darby canine kidney cells. Furthermore, alanine-scanning experiments using Lys40-containing peptides reveal that α-tubulin Ser38 is crucial for substrate recognition of αTAT1, whereas Asp39, Ile42, the glycine stretch (amino acid residues 43–45) and Asp46 are also involved. The requirement for substrate selection is totally different from that in various histone acetyltransferases, which appears to be consistent with the inability of αTAT1 to acetylate histones.

scholarly journals The functional dyad of scorpion toxin Pi1 is not itself a prerequisite for toxin binding to the voltage-gated Kv1.2 potassium channels

2004 ◽  
Vol 377 (1) ◽  
pp. 25-36 ◽  
Author(s):  
Stéphanie MOUHAT ◽  
Amor MOSBAH ◽  
Violeta VISAN ◽  
Heike WULFF ◽  
Muriel DELEPIERRE ◽  
...  
Keyword(s):  
Amino Acid ◽  
Scorpion Toxin ◽  
Xenopus Laevis Oocytes ◽  
Amino Acid Residues ◽  
Voltage Gated ◽  
Toxin Binding ◽  
Ic50 Values

Pi1 is a 35-residue scorpion toxin cross-linked by four disulphide bridges that acts potently on both small-conductance Ca2+-activated (SK) and voltage-gated (Kv) K+ channel subtypes. Two approaches were used to investigate the relative contribution of the Pi1 functional dyad (Tyr-33 and Lys-24) to the toxin action: (i) the chemical synthesis of a [A24,A33]-Pi1 analogue, lacking the functional dyad, and (ii) the production of a Pi1 analogue that is phosphorylated on Tyr-33 (P-Pi1). According to molecular modelling, this phosphorylation is expected to selectively impact the two amino acid residues belonging to the functional dyad without altering the nature and three-dimensional positioning of other residues. P-Pi1 was directly produced by peptide synthesis to rule out any possibility of trace contamination by the unphosphorylated product. Both Pi1 analogues were compared with synthetic Pi1 for bioactivity. In vivo, [A24,A33]-Pi1 and P-Pi1 are lethal by intracerebroventricular injection in mice (LD50 values of 100 and 40 µg/mouse, respectively). In vitro, [A24,A33]-Pi1 and P-Pi1 compete with 125I-apamin for binding to SK channels of rat brain synaptosomes (IC50 values of 30 and 10 nM, respectively) and block rat voltage-gated Kv1.2 channels expressed in Xenopus laevis oocytes (IC50 values of 22 µM and 75 nM, respectively), whereas they are inactive on Kv1.1 or Kv1.3 channels at micromolar concentrations. Therefore, although both analogues are less active than Pi1 both in vivo and in vitro, the integrity of the Pi1 functional dyad does not appear to be a prerequisite for the recognition and binding of the toxin to the Kv1.2 channels, thereby highlighting the crucial role of other toxin residues with regard to Pi1 action on these channels. The computed simulations detailing the docking of Pi1 peptides on to the Kv1.2 channels support an unexpected key role of specific basic amino acid residues, which form a basic ring (Arg-5, Arg-12, Arg-28 and Lys-31 residues), in toxin binding.

scholarly journals Role of Integrase in Reverse Transcription of the Saccharomyces cerevisiae Retrotransposon Ty1

2005 ◽  
Vol 4 (6) ◽  
pp. 1057-1065 ◽  
Author(s):  
M. Wilhelm ◽  
F.-X. Wilhelm
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Amino Acid ◽  
Specific Activity ◽  
Rnase H ◽  
Amino Acid Residues ◽  
Acidic Amino Acid ◽  
Acidic Domain ◽  
Basic Region

ABSTRACT Reverse transcriptase (RT) with its associated RNase H (RH) domain and integrase (IN) are key enzymes encoded by retroviruses and retrotransposons. Several studies have implied a functional role of the interaction between IN and RT during the replication of retroviral and retrotransposon genomes. In this study, IN deletion mutants were used to investigate the role of IN on the RT activity of the yeast Saccharomyces cerevisiae retrotransposon Ty1. We have identified two domains of Ty1 integrase which have effects on RT activity in vivo. The deletion of a domain spanning amino acid residues 233 to 520 of IN increases the exogenous specific activity of RT up to 20-fold, whereas the removal of a region rich in acidic amino acid residues between residues 521 and 607 decreases its activity. The last result complements our observation that an active recombinant RT protein can be obtained if a small acidic tail mimicking the acidic domain of IN is fused to the RT-RH domain. We suggest that interaction between these acidic amino acid residues of IN and a basic region of RT could be critical for the correct folding of RT and for the formation of an active conformation of the enzyme.

scholarly journals Dynamics of microtubules bundled by microtubule associated protein 2C (MAP2C).

1993 ◽  
Vol 120 (2) ◽  
pp. 451-465 ◽  
Author(s):  
T Umeyama ◽  
S Okabe ◽  
Y Kanai ◽  
N Hirokawa
Keyword(s):  
Amino Acid ◽  
Cdna Clone ◽  
Turnover Rate ◽  
Dynamic Properties ◽  
Nerve Cells ◽  
Amino Acid Residues ◽  
Abrupt Increase ◽  
Microtubule Networks ◽  
Rapid Incorporation

MAP2C is a microtubule-associated protein abundant in immature nerve cells. We isolated a cDNA clone encoding whole mouse MAP2C of 467 amino acid residues. In fibroblasts transiently transfected with cDNA of MAP2C, interphase microtubule networks were reorganized into microtubule bundles. To reveal the dynamic properties of microtubule bundles, we analyzed the incorporation sites of exogenously introduced tubulin by microinjection of biotin-labeled tubulin and the turnover rate of microtubule bundles by photoactivation of caged fluorescein-labeled tubulin. The injected biotin-labeled tubulin was rapidly incorporated into distal ends of preexisting microtubule bundles, suggesting a concentration of the available ends of microtubules at this region. Although homogenous staining of microtubule bundles with antibiotin antibody was observed 2 h after injection, the photoactivation study indicated that turnover of microtubule bundles was extremely suppressed and < 10% of tubulin molecules would be exchanged within 1 h. Multiple photoactivation experiments provided evidence that neither catastrophic disassembly at the distal ends of bundles nor concerted disassembly due to treadmilling at the proximal ends could explain the observed rapid incorporation of exogenously introduced tubulin molecules. We conclude that microtubules bundled by MAP2C molecules are very stable while the abrupt increase of free tubulin molecules by microinjection results in rapid assembly from the distal ends within the bundles as well as free nucleation of small microtubules which are progressively associated laterally with preexisting microtubule bundles. This is the first detailed study of the function of MAPs on the dynamics of microtubules in vivo.

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