scholarly journals Binding conformation and determinants of a single-chain peptide antagonist at the relaxin-3 receptor RXFP3

2018 ◽  
Vol 293 (41) ◽  
pp. 15765-15776 ◽  
Author(s):  
Linda M. Haugaard-Kedström ◽  
Han Siean Lee ◽  
Maryon V. Jones ◽  
Angela Song ◽  
Vishaal Rathod ◽  
...  
Keyword(s):  
Amino Acid ◽  
Helical Structure ◽  
Helical Conformation ◽  
Amino Acid Residues ◽  
Single Chain ◽  
Binding Conformation ◽  
Reward Seeking ◽  
Structure Activity ◽  
Relaxin Family ◽  
Key Driver

The neuropeptide relaxin-3 and its receptor relaxin family peptide receptor-3 (RXFP3) play key roles in modulating behavior such as memory and learning, food intake, and reward seeking. A linear relaxin-3 antagonist (R3 B1-22R) based on a modified and truncated relaxin-3 B-chain was recently developed. R3 B1-22R is unstructured in solution; thus, the binding conformation and determinants of receptor binding are unclear. Here, we have designed, chemically synthesized, and pharmacologically characterized more than 60 analogues of R3 B1-22R to develop an extensive understanding of its structure–activity relationships. We show that the key driver for affinity is the nonnative C-terminal Arg23. Additional contributors to binding include amino acid residues that are important also for relaxin-3 binding, including Arg12, Ile15, and Ile19. Intriguingly, amino acid residues that are not exposed in native relaxin-3, including Phe14 and Ala17, also interact with RXFP3. We show that R3 B1-22R has a propensity to form a helical structure, and modifications that support a helical conformation are functionally well-tolerated, whereas helix breakers such as proline residues disrupt binding. These data suggest that the peptide adopts a helical conformation, like relaxin-3, upon binding to RXFP3, but that its smaller size allows it to penetrate deeper into the orthosteric binding site, creating more extensive contacts with the receptor.

scholarly journals Primary- and secondary-structural analysis of a unique prokaryotic metallothionein from a Synechococcus sp. cyanobacterium

1988 ◽  
Vol 251 (3) ◽  
pp. 691-699 ◽  
Author(s):  
R W Olafson ◽  
W D McCubbin ◽  
C M Kay
Keyword(s):  
Amino Acid ◽  
Metal Binding ◽  
Helical Structure ◽  
Basic Amino Acid ◽  
Cysteine Residues ◽  
Amino Acid Residues ◽  
C Terminus ◽  
Empirical Relations ◽  
Heavy Metal Binding ◽  
Synechococcus Sp

Biochemical and physiological studies of Synechococcus cyanobacteria have indicated the presence of a low-Mr heavy-metal-binding protein with marked similarity to eukaryotic metallothioneins (MTs). We report here the characterization of a Synechococcus prokaryotic MT isolated by gel-permeation and reverse-phase chromatography. The large number of variants of this molecule found during chromatographic separation could not be attributed to the presence of major isoproteins as assessed by amino acid analysis and amino acid sequencing of isoforms. Two of the latter were shown to have identical primary structures that differed substantially from the well-described eukaryotic MTs. In addition to six long-chain aliphatic residues, two aromatic residues were found adjacent to one another near the centre of the molecule, making this the most hydrophobic MT to be described. Other unusual features included a pair of histidine residues located in repeating Gly-His-Thr-Gly sequences near the C-terminus and a complete lack of association of hydroxylated residues with cysteine residues, as is commonly found in eukaryotes. Similarly, aside from a single lysine residue, no basic amino acid residues were found adjacent to cysteine residues in the sequence. Most importantly, sequence alignment analyses with mammalian, invertebrate and fungal MT sequences showed no statistically significant homology aside from the presence of Cys-Xaa-Cys structures common to all MTs. On the other hand, like other MTs, the prokaryotic molecule appears to be free of alpha-helical structure but has a considerable amount of beta-structure, as predicted by both c.d. measurements and the Chou & Fasman empirical relations. Considered together, these data suggested that some similarity between the metal-thiolate clusters of the prokaryote and eukaryote MTs may exist.

scholarly journals Development of Antimicrobial Stapled Peptides Based on Magainin 2 Sequence

Molecules ◽  
2021 ◽  
Vol 26 (2) ◽  
pp. 444
Author(s):  
Motoharu Hirano ◽  
Chihiro Saito ◽  
Hidetomo Yokoo ◽  
Chihiro Goto ◽  
Ryuji Kawano ◽  
...  
Keyword(s):  
Antimicrobial Activity ◽  
Amino Acid ◽  
Hemolytic Activity ◽  
Helical Structure ◽  
Antimicrobial Activities ◽  
Side Chain ◽  
Membrane Disruption ◽  
Amino Acid Residues ◽  
Electrophysiological Measurements ◽  
Cell Membrane Disruption

Magainin 2 (Mag2), which was isolated from the skin of the African clawed frog, is a representative antimicrobial peptide (AMP) that exerts antimicrobial activity via microbial membrane disruption. It has been reported that the helicity and amphipathicity of Mag2 play important roles in its antimicrobial activity. We investigated and recently reported that 17 amino acid residues of Mag2 are required for its antimicrobial activity, and accordingly developed antimicrobial foldamers containing α,α-disubstituted amino acid residues. In this study, we further designed and synthesized a set of Mag2 derivatives bearing the hydrocarbon stapling side chain for helix stabilization. The preferred secondary structures, antimicrobial activities, and cell-membrane disruption activities of the synthesized peptides were evaluated. Our analyses revealed that hydrocarbon stapling strongly stabilized the helical structure of the peptides and enhanced their antimicrobial activity. Moreover, peptide 2 stapling between the first and fifth position from the N-terminus showed higher antimicrobial activity than that of Mag2 against both gram-positive and gram-negative bacteria without exerting significant hemolytic activity. To investigate the modes of action of tested peptides 2 and 8 in antimicrobial and hemolytic activity, electrophysiological measurements were performed.

scholarly journals The Primary Structure of Antithrombin-III

1977 ◽  
Author(s):  
T. E. Petersen ◽  
G. Dudek-Wojciechowska ◽  
L. Sottrup-Jensen ◽  
S. Magnusson
Keyword(s):  
Amino Acid ◽  
Antithrombin Iii ◽  
Factor Xa ◽  
Amino Acid Residues ◽  
Disulfide Bridges ◽  
Single Chain ◽  
Terminal Sequence ◽  
Chymotryptic Peptide ◽  
Sequence Region ◽  
Bovine Factor

Human antithrombin-III is a single-chain glycoprotein with three disulfide bridges and four prosthetic glucosamine-based oligosaccharide groups. The disulfide bridges have been established. In four fragments of 208, 168, 3 and 46 amino acid residues, resp. 415 of the appr. 425 residues have been sequenced. The four oligosaccharide groups are attached to four Asn-residues within a sequence region of 95 residues. No extensive sequence homology with the trypsin inhibitors has been observed. One chymotryptic peptide was found to be a substrate for bovine factor Xa, cleaving the arginyl bond in the sequence -Ile-Val-Ala-Glu-Gly-Arg-Asp-. A second peptide is cleaved by thrombin. It is not clear whether these sites are inhibitor sites in the native molecule. Other possible candidates for inhibitor sites are a -Val-Leu-Ile-Leu-Pro-Lys-Pro- sequence (similar to the sequence 40-48 of hirudin, which also includes a -Pro-Lys-Pro- sequence) and also the C-terminal sequence -Gly-Arg-Val-Ala-Asn-Pro-Cys-Val-Lys.

Structure-activity studies of dermorphin. The role of side chains of amino acid residues on the biological activity of dermorphin

Peptides ◽  
1984 ◽  
Vol 5 (4) ◽  
pp. 687-689 ◽  
Author(s):  
Krzysztof Darłak ◽  
Zbigniew Grzonka ◽  
Pawel Krzaścik ◽  
Piotr Janicki ◽  
S.Witold Gumułka
Keyword(s):  
Biological Activity ◽  
Amino Acid ◽  
Side Chains ◽  
Amino Acid Residues ◽  
Structure Activity

scholarly journals The amino acid sequence of γ-crystallin (fraction II) from calf lens

1972 ◽  
Vol 128 (4) ◽  
pp. 961-970 ◽  
Author(s):  
L. R. Croft
Keyword(s):  
Amino Acid ◽  
Amino Acid Sequence ◽  
Amino Acid Residues ◽  
Single Chain

The amino acid sequence of γ-crystallin (fraction II) from calf lens has been determined; this indicates it to be a single-chain polypeptide of 165 amino acid residues.

scholarly journals Increasing protein stability by engineering the n → π* interaction at the β-turn

2020 ◽  
Vol 11 (35) ◽  
pp. 9480-9487 ◽  
Author(s):  
Bhavesh Khatri ◽  
Puja Majumder ◽  
Jayashree Nagesh ◽  
Aravind Penmatsa ◽  
Jayanta Chatterjee
Keyword(s):  
Amino Acid ◽  
Protein Stability ◽  
Structural Stability ◽  
Direct Consequence ◽  
Helical Conformation ◽  
Amino Acid Residues ◽  
Π Interactions

Amino acid residues adopt a right-handed α-helical conformation with increasing strength of the n → π* interaction. We also demonstrate a direct consequence of n → π* interactions on enhancing the structural stability of proteins.

scholarly journals Amino acid sequence of versutoxin, a lethal neurotoxin from the venom of the funnel-web spider Atrax versutus

1988 ◽  
Vol 250 (2) ◽  
pp. 401-405 ◽  
Author(s):  
M R Brown ◽  
D D Sheumack ◽  
M I Tyler ◽  
M E H Howden
Keyword(s):  
Amino Acid ◽  
Amino Acid Sequence ◽  
Gas Phase ◽  
Edman Degradation ◽  
Amino Acid Residues ◽  
Single Chain ◽  
Male And Female ◽  
Web Spider ◽  
Phase Protein ◽  
Protein Sequencer

The complete amino acid sequence of versutoxin, a lethal neurotoxic polypeptide isolated from the venom of male and female funnel-web spiders of the species Atrax versutus, was determined. Sequencing was performed in a gas-phase protein sequencer by automated Edman degradation of the S-carboxymethylated toxin and fragments of it produced by reaction with CNBr. Versutoxin consisted of a single chain of 42 amino acid residues. It was found to have a high proportion of basic residues and of cystine. The primary structure showed marked homology with that of robustoxin, a novel neurotoxin recently isolated from the venom of another funnel-web-spider species, Atrax robustus.

Molecular analysis of human anti-factor VIII antibodies by V gene phage display identifies a new epitope in the acidic region following the A2 domain

Blood ◽  
2000 ◽  
Vol 96 (2) ◽  
pp. 540-545 ◽  
Author(s):  
Edward N. van den Brink ◽  
Ellen A. M. Turenhout ◽  
Christine M. C. Bank ◽  
Karin Fijnvandraat ◽  
Marjolein Peters ◽  
...  
Keyword(s):  
Amino Acid ◽  
Phage Display ◽  
Factor Viii ◽  
Gene Segment ◽  
Amino Acid Residues ◽  
Germline Gene ◽  
Single Chain ◽  
Factor Viii Activity ◽  
A2 Domain ◽  
Acidic Region

One of the major binding sites for factor VIII inhibitors is located within the A2 domain. In this study, phage display technology was used to isolate 2 human monoclonal antibodies, termed VK34 and VK41, directed toward the heavy chain of factor VIII. The VHdomain of a single-chain variable domain antibody fragment (scFv) VK34 is encoded by germline gene segment DP-10. Epitope-mapping studies revealed that scFv VK34 is directed against amino acid residues Arg484–Ile508 , a previously identified binding site for factor VIII inhibitors in the A2 domain. ScFv VK34 inhibited factor VIII activity with a titer of 280 BU/mg. The VH domain of VK41 was encoded by germline gene segment DP-47. A phage corresponding to VK41 competed with a monoclonal antibody for binding to amino acid residues Asp712–Ala736 in the acidic region adjacent to the A2 domain. Reactivity of VK41 with a factor VIII variant in which we replaced amino acid residues Asp712–Ala736for the corresponding region of heparin cofactor II was strongly reduced. In addition, substitution of Tyr718719723 for Phe abrogated binding of VK41 to factor VIII. ScFv VK41 did not inhibit factor VIII activity. This study not only defines the primary structure of human anti-factor VIII antibodies reactive with the A2 domain, it also describes an antibody with an epitope not previously identified in the antibody repertoire of hemophilia patients with an inhibitor.

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