scholarly journals Positive Charges of Translocating Polypeptide Chain Retrieve an Upstream Marginal Hydrophobic Segment from the Endoplasmic Reticulum Lumen to the Translocon

2010 ◽  
Vol 21 (12) ◽  
pp. 2045-2056 ◽  
Author(s):  
Hidenobu Fujita ◽  
Yuichiro Kida ◽  
Masatoshi Hagiwara ◽  
Fumiko Morimoto ◽  
Masao Sakaguchi
Keyword(s):  
Endoplasmic Reticulum ◽  
Amino Acid ◽  
Membrane Proteins ◽  
Polypeptide Chain ◽  
Water Environment ◽  
Amino Acid Residues ◽  
Endoplasmic Reticulum Lumen ◽  
Membrane Spanning ◽  
Positively Charged

Positively charged amino acid residues are well recognized topology determinants of membrane proteins. They contribute to the stop-translocation of a polypeptide translocating through the translocon and to determine the orientation of signal sequences penetrating the membrane. Here we analyzed the function of these positively charged residues during stop-translocation in vitro. Surprisingly, the positive charges facilitated membrane spanning of a marginally hydrophobic segment, even when separated from the hydrophobic segment by 70 residues. In this case, the hydrophobic segment was exposed to the lumen, and then the downstream positive charges triggered the segment to slide back into the membrane. The marginally hydrophobic segment spanned the membrane, but maintained access to the water environment. The positive charges not only fix the hydrophobic segment in the membrane at its flanking position, but also have a much more dynamic action than previously realized.

Translocation of neutral endopeptidase 24.11 mutants with deletions of the NH2- terminal cytosolic domain

1994 ◽  
Vol 72 (5-6) ◽  
pp. 182-187
Author(s):  
Isabelle Lemire ◽  
Patrice Roy ◽  
Guy Boileau
Keyword(s):  
Amino Acid ◽  
Membrane Proteins ◽  
Amino Acid Residue ◽  
Polypeptide Chain ◽  
Neutral Endopeptidase ◽  
Terminal Domain ◽  
Endopeptidase 24.11 ◽  
Cytosolic Domain ◽  
Microsomal Membranes ◽  
Positively Charged

Rabbit neutral endopeptidase 24.11 (NEP) is a type II membrane protein with a positively charged 27 amino acid residue NH2-terminal cytoplasmic domain, a 20 amino acid residue hydrophobic signal peptide/membrane anchor domain, and a large catalytic COOH-terminal domain exposed on the exoplasmic side of the membrane. To study the role of the cytosolic domain in anchoring NEP in the plasma membrane, we constructed two mutants in which this cytosolic domain was deleted. In the first mutant (NEPΔcyto), a Glu residue was present in NH2-terminus, while a Lys residue was substituted at the same position in the second mutant (NEPΔcyto(K)). To better understand the interaction of these mutants with the rough endoplasmic reticulum membrane, the mutated NEP cDNAs were transcribed and translated in vitro in the presence of microsomal membranes. Our studies showed that deletion of the hydrophillic cytosolic domain affects translocation of the NEP polypeptide chain. Substitution of a positively charged Lys residue for the Glu residue at the NH2-terminus of the deletion mutant only partly restored translocation of the polypeptide chain. Furthermore, carbonate extraction and trypsin digestion of the microsomal membranes indicated that the deletion mutants are inserted in the microsomal membranes as type III membrane proteins with their COOH-terminal domain exposed on the exterior of the microsomes. Thus, efficient translocation is dependent on the presence of a charged cytoplasmic domain.Key words: membrane proteins, translocation, cytosolic domain, neutral endopeptidase 24.11.

scholarly journals Saccharomyces cerevisiae Rhodanese RDL2 Uses the Arg Residue of the Active-Site Loop for Thiosulfate Decomposition

Antioxidants ◽  
2021 ◽  
Vol 10 (10) ◽  
pp. 1525
Author(s):  
Qingda Wang ◽  
Huanjie Li ◽  
Yongzhen Xia ◽  
Luying Xun ◽  
Huaiwei Liu
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Amino Acid ◽  
Active Site ◽  
In Vitro Assays ◽  
Amino Acid Residues ◽  
Sulfane Sulfur ◽  
Sulfur Transfer ◽  
Sulfur Containing ◽  
Positively Charged

Persulfide, polysulfide and thiosulfate are examples of sulfane sulfur containing chemicals that play multiple functions in biological systems. Rhodaneses are widely present in all three kingdoms of life, which catalyze sulfur transfer among these sulfane sulfur-containing chemicals. The mechanism of how rhodaneses function is not well understood. Saccharomyces cerevisiae rhodanese 2 (RDL2) is involved in mitochondrial biogenesis and cell cycle control. Herein, we report a 2.47 Å resolution structure of RDL2 co-crystallized with thiosulfate (PDB entry: 6K6R). The presence of an extra sulfur atom Sδ, forming a persulfide bond with the Sγ atom of Cys106, was observed. Distinct from the persulfide groups in GlpE (PDB entry:1GMX) and rhobov (PDB entry:1BOI), the persulfide group of RDL2 is located in a peanut-like pocket of the neutral electrostatic field and is far away from positively charged amino acid residues of its active-site loop, suggesting no interaction between them. This finding suggests that the positively charged amino acid residues are not involved in the stabilization of the persulfide group. Activity assays indicate that the Arg111 of the active-site loop is critical for the sulfane sulfur transfer. In vitro assays indicate that Arg propels the thiosulfate decomposition. Thus, we propose that Arg can offer a hydrogen bond-rich, acidic-like microenvironment in RDL2 in which thiosulfate decomposes to release sulfane sulfur. Thr of the active-site loop of rhodaneses has the same functions as Arg. Our proposal may explain the catalyzing mechanism of rhodaneses.

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.

Amino acid sequence of cyanogen bromide fragment CB5 of human hemopexin

1989 ◽  
Vol 54 (3) ◽  
pp. 803-810 ◽  
Author(s):  
Ivan Kluh ◽  
Ladislav Morávek ◽  
Manfred Pavlík
Keyword(s):  
Amino Acid ◽  
Amino Acid Sequence ◽  
Acid Hydrolysis ◽  
Cyanogen Bromide ◽  
Polypeptide Chain ◽  
Amino Acid Residues ◽  
Mild Acid Hydrolysis ◽  
Methionine Residue ◽  
Cyanogen Bromide Fragment ◽  
Mild Acid

Cyanogen bromide fragment CB5 represents the region of the polypeptide chain of hemopexin between the fourth and fifth methionine residue (residues 232-352). It contains 120 amino acid residues in the following sequence: Arg-Cys-Ser-Pro-His-Leu-Val-Leu-Ser-Ala-Leu-Thr-Ser-Asp-Asn-His-Gly-Ala-Thr-Tyr-Ala-Phe-Ser-Gly-Thr-His-Tyr-Trp-Arg-Leu-Asp-Thr-Ser-Arg-Asp-Gly-Trp-His-Ser-Trp-Pro-Ile-Ala-His-Gln-Trp-Pro-Gln-Gly-Pro-Ser-Ala-Val-Asp-Ala-Ala-Phe-Ser-Trp-Glu-Glu-Lys-Leu-Tyr-Leu-Val-Gln-Gly-Thr-Gln-Val-Tyr-Val-Phe-Leu-Thr-Lys-Gly-Gly-Tyr-Thr-Leu-Val-Ser-Gly-Tyr-Pro-Lys-Arg-Leu-Glu-Lys-Glu-Val-Gly-Thr-Pro-His-Gly-Ile-Ile-Leu-Asp-Ser-Val-Asp-Ala-Ala-Phe-Ile-Cys-Pro-Gly-Ser-Ser-Arg-Leu-His-Ile-Met. The sequence was derived from the data on peptides prepared by cleavage of fragment CB5 by mild acid hydrolysis, by trypsin and chymotrypsin.

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).

Engineering two mutants of cDNA-encoding G2 subunit of soybean glycinin capable of self-assembly in vitro and rich in methionine

Biologia ◽  
2007 ◽  
Vol 62 (4) ◽  
Author(s):  
Reda Sammour
Keyword(s):  
Amino Acid ◽  
Self Assembly ◽  
Seed Quality ◽  
Amino Acid Residues ◽  
Methionine Residues

AbstractThe main goal of this work was to make the cDNA-encoding subunit G2 of soybean glycinin, capable of self-assembly in vitro and rich in methionine residues. Two mutants (pSP65/G4SacG2 and pSP65/G4SacG2HG4) were therefore constructed. The constructed mutants were successfully assembled in vitro into oligomers similar to those occurred in the seed. The successful self-assembly was due to the introduction of Sac fragment of Gy4 (the codons of the first 21 amino acid residues), which reported to be the key element in self-assembly into trimers. The mutant pSP65/G4SacG2HG4 included the acidic chain of Gy4 (HG4), which was previously molecularly modified to have three methionine residues. This mutant will be useful in the efforts to improve the seed quality.

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