scholarly journals Molecular Dissection of dH3w, A Fluorescent Peptidyl Sensor for Zinc and Mercury

Sensors ◽  
2020 ◽  
Vol 20 (3) ◽  
pp. 598 ◽  
Author(s):  
Marialuisa Siepi ◽  
Rosario Oliva ◽  
Filomena Battista ◽  
Luigi Petraccone ◽  
Pompea Del Vecchio ◽  
...  
Keyword(s):  
Tryptophan Residue ◽  
Histidine Residues ◽  
Turn On ◽  
Dansyl Group ◽  
C Terminus ◽  
Molecular Behavior ◽  
Fluorescent Peptide ◽  
Acetyl Group ◽  
High Concentrations ◽  
Fluorescent Moiety

Previously, we reported that fluorescent peptide dansyl-HPHGHW-NH2 (dH3w), designed on the repeats of the human histidine-rich glycoprotein, shows a turn-on response to Zn(II) and a complex response to Hg(II) characterized by a turn-off phase at low Hg(II) concentrations and a turn-on phase at high concentrations. As Hg(II) easily displaces Zn(II), dH3w is a useful probe for the environmental monitoring of Hg(II). In order to investigate the molecular basis of the metal selectivity and fluorescence response, we characterized three variants, dH3w(H1A), dH3w(H3A), and dH3w(H5A), in which each of the three histidine residues was changed to alanine, and two variants with a single fluorescent moiety, namely dH3w(W6A), in which the tryptophan residue at the C-terminus was changed to alanine, and AcH3w, in which the N-terminal dansyl moiety was substituted by an acetyl group. These variants allowed us to demonstrate that all the histidine residues are essential for a strong interaction with Zn(II), whereas two histidine residues (in particular His5) and the dansyl group are necessary to bind Hg(II). The data reported herein shed light on the molecular behavior of dH3w, thus paving the way to the rational designing of further and more efficient fluorescent peptidyl probes for Hg(II).

scholarly journals External Nickel Inhibits Epithelial Sodium Channel by Binding to Histidine Residues within the Extracellular Domains of α and γ Subunits and Reducing Channel Open Probability

2002 ◽  
Vol 277 (51) ◽  
pp. 50098-50111 ◽  
Author(s):  
Shaohu Sheng ◽  
Clint J. Perry ◽  
Thomas R. Kleyman
Keyword(s):  
Divalent Cations ◽  
Wild Type Mouse ◽  
Inhibitory Effect ◽  
Inward Rectification ◽  
Dependent Manner ◽  
Open Probability ◽  
Histidine Residues ◽  
Low Concentrations ◽  
High Concentrations ◽  
A Site

Epithelial sodium channels (ENaC) are regulated by various intracellular and extracellular factors including divalent cations. We studied the inhibitory effect and mechanism of external Ni2+on cloned mouse α-β-γ ENaC expressed inXenopusoocytes. Ni2+reduced amiloride-sensitive Na+currents of the wild type mouse ENaC in a dose-dependent manner. The Ni2+block was fast and partially reversible at low concentrations and irreversible at high concentrations. ENaC inhibition by Ni2+was accompanied by moderate inward rectification at concentrations higher than 0.1 mm. ENaC currents were also blocked by the histidine-reactive reagent diethyl pyrocarbonate. Pretreatment of the oocytes with the reagent reduced Ni2+inhibition of the remaining current. Mutations at αHis282and γHis239located within the extracellular loops significantly decreased Ni2+inhibition of ENaC currents. The mutation αH282D or double mutations αH282R/γH239R eliminated Ni2+block. All mutations at γHis239eliminated Ni2+-induced inward current rectification. Ni2+block was significantly enhanced by introduction of a histidine at αArg280. Lowering extracellular pH to 5.5 and 4.4 decreased or eliminated Ni2+block. Although αH282C-β-γ channels were partially inhibited by the sulfhydryl-reactive reagent [2-(trimethylammonium)ethyl] methanethiosulfonate bromide (MTSET), α-β-γ H239C channels were insensitive to MTSET. From patch clamp studies, Ni2+did not affect unitary current but decreased open probability when perfused into the recording pipette. Our results suggest that external Ni2+reduces ENaC open probability by binding to a site consisting of αHis282and γHis239and that these histidine residues may participate in ENaC gating.

scholarly journals Investigation of human erythrocyte superoxide dismutase by 1H nuclear-magnetic-resonance spectroscopy

1980 ◽  
Vol 185 (1) ◽  
pp. 245-252 ◽  
Author(s):  
H A O Hill ◽  
W K Lee ◽  
J V Bannister ◽  
W H Bannister
Keyword(s):  
Superoxide Dismutase ◽  
Active Site ◽  
Human Erythrocyte ◽  
Superoxide Dismutases ◽  
Resonance Spectroscopy ◽  
Structural Homology ◽  
Histidine Residues ◽  
Erythrocyte Superoxide Dismutase ◽  
1H Nuclear Magnetic Resonance ◽  
Acetyl Group

The 170MHZ 1 H n.m.r. spectra of the Cu(II)/Zn(II), Cu(I)/Zn(II) and apo- forms of human erythrocyte superoxide dismutase (EC 1.15.1.1) are reported. Resonances are assigned to the C-2 and C-4 protons of histidine residues in the active site, and it is suggested that five or six histidine residues serve as ligands to the metal ions in each subunit of the enzyme. The remaining assigned resonances are associated with histidine-41, N-terminal N-acetyl group, histidine- 108 and cysteine- 109. A comparison of the n.m.r. spectra of human and bovine superoxide dismutases suggests significant structural homology.

scholarly journals Systematic Analysis of a Conserved Region of the Aminoglycoside 6′-N-Acetyltransferase Type Ib

2001 ◽  
Vol 45 (12) ◽  
pp. 3287-3292 ◽  
Author(s):  
Ali Shmara ◽  
Natalia Weinsetel ◽  
Ken J. Dery ◽  
Ramona Chavideh ◽  
Marcelo E. Tolmasky
Keyword(s):  
Amino Acids ◽  
Active Form ◽  
Major Change ◽  
Cysteine Residue ◽  
Alanine Scanning Mutagenesis ◽  
Systematic Analysis ◽  
C Terminus ◽  
Acetyl Group ◽  
Semisynthetic Derivative ◽  
High Level

ABSTRACT Alanine-scanning mutagenesis was applied to the aminoglycoside 6′-N-acetyltransferase type Ib conserved motif B, and the effects of the substitutions were analyzed by measuring the MICs of kanamycin (KAN) and its semisynthetic derivative, amikacin (AMK). Several substitutions resulted in no major change in MICs. E167A and F171A resulted in derivatives that lost the ability to confer resistance to KAN and AMK. P155A, P157A, N159A, L160A, I163A, K168A, and G170A conferred intermediate levels of resistance. Y166A resulted in an enzyme derivative with a modified specificity; it conferred a high level of resistance to KAN but lost the ability to confer resistance to AMK. Although not as pronounced, the resistance profiles conferred by substitutions N159A and G170A were related to that conferred by Y166A. These phenotypes, taken together with previous results indicating that mutant F171L could not catalyze acetylation of AMK when the assays were carried out at 42°C (D. Panaite and M. Tolmasky, Plasmid 39:123–133, 1998), suggest that some motif B amino acids play a direct or indirect role in acceptor substrate specificity. MICs of AMK and KAN for cells harboring the substitution C165A were high, suggesting that the active form of the enzyme may not be a dimer formed through a disulfide bond. Furthermore, this result indicated that the acetylation reaction occurs through a direct mechanism rather than a ping-pong mechanism that includes a transient transfer of the acetyl group to a cysteine residue. Deletion of fragments at the C terminus demonstrated that up to 10 amino acids could be deleted without a loss of activity.

scholarly journals Unique translational modification of an invertebrate neuropeptide: a phosphorylated member of the adipokinetic hormone peptide family

2006 ◽  
Vol 393 (3) ◽  
pp. 705-713 ◽  
Author(s):  
Gerd Gäde ◽  
Petr Šimek ◽  
Kevin D. Clark ◽  
Lutz Auerswald
Keyword(s):  
Tryptophan Fluorescence ◽  
Complete Sequence ◽  
Single Step ◽  
Post Translational Modification ◽  
Adipokinetic Hormone ◽  
Multi Stage ◽  
C Terminus ◽  
Peptide Family ◽  
Natural Peptide ◽  
High Concentrations

Separation of an extract of corpora cardiaca from the protea beetle, Trichostetha fascicularis, by single-step RP (reverse-phase)-HPLC and monitoring of tryptophan fluorescence resulted in two distinctive peaks, the material of which mobilized proline and carbohydrates in a bioassay performed using the beetle. Material from one of these peaks was; however, inactive in the classical bioassays of locusts and cockroaches that are used for detecting peptides belonging to the AKH (adipokinetic hormone) family. After enzymatically deblocking the N-terminal pyroglutamic acid (pGlu) residue in the peptide material and sequencing by Edman degradation, a partial sequence was obtained: (pGlu)-Ile-Asn-Met-Thr-Xaa-Gly-Trp. The complete sequence was deduced from ESI-MSn (electrospray ionization multi-stage-MS); position six was identified as a phosphothreonine residue and the C-terminus is amidated. The peptide, code-named Trifa-CC, was chemically synthesized and used in confirmatory experiments to show that the primary structure had been correctly assigned. To our knowledge, this is the first report of a phosphorylated invertebrate neuropeptide. Synthetic Trifa-CC co-elutes with the natural peptide, found in the gland of the protea beetle, after RP-HPLC. Moreover, the natural peptide can be dephosphorylated by alkaline phosphatase and the product of that reaction has the same retention time as a synthetic nonphosphorylated octapeptide which has the same sequence as Trifa-CC. Finally, synthetic Trifa-CC has hypertrehalosaemic and hyperprolinaemic biological activity in the protea beetle, but even high concentrations of synthetic Trifa-CC are inactive in locusts and cockroaches. Hence, the correct peptide structure has been assigned. Trifa-CC of the protea beetle is an unusual member of the AKH family that is unique in its post-translational modification. Since it increases the concentration of carbohydrates and proline in the haemolymph when injected into the protea beetle, and since these substrates are also used during flight, we hypothesize that Trifa-CC controls the mobilization of these metabolites in the protea beetle.

scholarly journals A computational study of Trishomocubane amino acid dipeptide

2004 ◽  
Author(s):  
◽  
Poomani Penny Govender
Keyword(s):  
Amino Acid ◽  
Computational Study ◽  
Building Blocks ◽  
Conformational Space ◽  
Basis Set ◽  
Peptide Design ◽  
Helical Structures ◽  
Hartree Fock ◽  
C Terminus ◽  
Acetyl Group

4-amino-(D3)-trishomocubane-4-carboxylic acid (tris-amino acid) is a constrained a-amino acid residue that exhibits peculiar conformational characteristics. The aim of the present study is to provide a deeper understanding of these features, which can be used as a guide when chOOSing@shomocubane as suitable building blocks for peptide design. The Ca carbon of@ishomocubane forms part of the cyclic structure, and consequently a peptidic environment was simulated with an acetyl group on its N-terminus and a methyl amide group on its C-terminus. This study involved a complete exploration of the conformational profile of (Yishomocubane using computational techniques.The parm94 parametization of the AMBER oio forc@eld was used to explore the conformational space of the peptide,Q)\xEFshomocubane. The Ramachandran maps computed at the molecular mechanics level' with the parm94 forc@\xEFeld parameters compared reasonably with the corresponding maps computed at the Hartree Fock (HF) level, using the 6-31G* basis set. The results of this study revealed that the conformational profile of the @ishomocubane peptide can be characterized by four low energy regions, viz., C7ax, C7eq, 310 and al helical structures.

Calcium Ion Binding Within Fibrinogen Fragment D

1981 ◽  
Author(s):  
David W Britton ◽  
Jan S Lawrie ◽  
Graham D Kemp
Keyword(s):  
Calcium Ions ◽  
Calcium Ion ◽  
Cross Linking ◽  
C Terminus ◽  
Dimethyl Suberimidate ◽  
High Concentrations ◽  
The Stability ◽  
Considerable Body ◽  
Chemical Cross Linking ◽  
Compact Conformation

Fibrinogen contains a number of strongly bound calcium. ions and a considerable body of evidence new exists to show that the plasmin degradation product fragment D contains one strongly bound calciumion. It is also established that this calcium ion has a notable effect on the plasmin resistance of the molecule. Previous work from this laboratory strongly suggests that the binding site is located towards the C-terminus of the γ chain. We have also investigated the influence of calcium. ions on the conformation of fragment D by ultracentrifugation and chemical cross-linking. In the presence of calcium ions there is a preponderance of intra-molecular cross-linking even at high concentrations of fragment D using bisimidates such as dimethyl suberimidate and dimethyl adipimidate. In the absence of calcium. ions there is an increase in the extent of inter-molecular cross-linking. From such evidence we would propose that calcium ions stabilise a compact conformation within fragment D. The presence of calcium ions also affects the stability of the D:E complex.

A radical approach for fluorescent turn ‘on’ detection, differentiation and bioimaging of methanol

2015 ◽  
Vol 13 (33) ◽  
pp. 8822-8826 ◽  
Author(s):  
Virendra Kumar ◽  
Ajit Kumar ◽  
Uzra Diwan ◽  
Manish Kumar Singh ◽  
K. K. Upadhyay
Keyword(s):  
Schiff Base ◽  
Control Unit ◽  
Selective Detection ◽  
Fluorescent Material ◽  
Turn On ◽  
Cyclic Control ◽  
Fluorescent Moiety ◽  
Radical Approach

A Schiff base (RC) is presented herein as a smart fluorescent material for the selective detection and bioimaging of methanol. The key step behind same involves methanol induced opening of the cyclic control unit ofRCresulting in the formation of a highly fluorescent moiety,RO.

Amino Acid Sequence Studies of Horseradish Peroxidase. II. Thermolytic Peptides

1972 ◽  
Vol 50 (1) ◽  
pp. 63-90 ◽  
Author(s):  
K. G. Welinder ◽  
L. B. Smillie
Keyword(s):  
Amino Acid ◽  
Horseradish Peroxidase ◽  
Amino Acid Sequence ◽  
Cation Exchanger ◽  
Paper Electrophoresis ◽  
Tryptophan Residue ◽  
Amino Acid Residues ◽  
Disulfide Bridges ◽  
Serine Residue ◽  
C Terminus

Horseradish peroxidase (HRP) was digested with thermolysin. On fractionation on Sephadex G-25, Fine Chromobeads type P (Dowex 50 type resin) and by high-voltage paper electrophoresis, we isolated about 120 thermolytic peptides. Some experimentation on the composition of the pyridine acetate gradient, used for elution of the cation exchanger, is reported. All peptides were characterized with respect to amino acid composition, N-terminal residue, and pH 6.5 mobility. Unknown peptides or peptides not corresponding unambiguously to previously established tryptic sequences were subjected to dansyl-Edman analysis. Thermolytic peptides accounting for all tryptic sequences except a dipeptide and a tripeptide, and unique thermolytic sequences accounting for about 100 amino acid residues, were obtained. Nine convincing and several indicative overlaps were established for known tryptic sequences. The sequences around all four disulfide bridges, the three histidine residues, and the only tryptophan residue have been elucidated. Eight sites of carbohydrate attachment have been identified. For seven of these sites we have evidence for attachment to asparagine, and for six of the sites the carbohydrate-bound asparagine was found in the well-known sequences Asn–X–Ser/Thr. The remaining two sequences, though incomplete, are compatible with this pattern. Tentatively we suggest a pyrrolidone carboxyl N-terminal for HRP. The specificity of trypsin implicates a sequence found in two varieties, differing only by a C-terminal serine residue at the C-terminus of HRP. A discussion of the possible complications of the acidic heme extraction on the results obtained is included.

scholarly journals Pitfalls in Diagnostic Gastrin Measurements

2012 ◽  
Vol 58 (5) ◽  
pp. 831-836 ◽  
Author(s):  
Jens F Rehfeld ◽  
Linda Bardram ◽  
Linda Hilsted ◽  
Pierre Poitras ◽  
Jens P Goetze
Keyword(s):  
Plasma Proteins ◽  
Clinical Chemistry ◽  
Diagnostic Sensitivity ◽  
Delay In Diagnosis ◽  
Peptide Pattern ◽  
C Terminus ◽  
Low Concentrations ◽  
Lethal Complications ◽  
High Concentrations ◽  
Commercial Kits

Abstract BACKGROUND Gastrin measurements are performed primarily for the diagnosis of gastrin-producing tumors, gastrinomas, which cause the Zollinger–Ellison syndrome (ZES). Gastrin circulates as several bioactive peptides, however, and the peptide pattern in gastrinoma patients often deviates from normal. Therefore, it is necessary to measure all forms of gastrin. CONTENT Only immunoassays are useful for measurement of gastrin in plasma. The original assays were RIAs developed in research laboratories that used antibodies directed against the C terminus of gastrin peptides. Because the C-terminal tetrapeptide amide sequence constitutes the active site of gastrin peptides, these assays were well suited for gastrinoma diagnosis. More recently, however, most clinical chemistry laboratories have switched to commercial kits. Because of recent cases of kit-measured normogastrinemia in patients with ZES symptoms, the diagnostic sensitivity and analytical specificity of the available kits have been examined. The results show that gastrin kits frequently measure falsely low concentrations because they measure only a single gastrin form. Falsely high concentrations were also encountered, owing to overreactivity with O-sulfated gastrins or plasma proteins. Thus, more than half of the gastrin kits on the market are unsuited for diagnostics. SUMMARY Gastrinomas are neuroendocrine tumors, some of which become malignant. A delay in diagnosis leads to fulminant ZES, with major, even lethal, complications. Consequently, it is necessary that the diagnostic sensitivity of gastrin kits be adequate. This diagnostic sensitivity requires antibodies that bind the C-terminal epitope of bioactive gastrins without the influence of O-sulfation.

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