Defining the familial fold of the vicilin-buried peptide family

ABSTRACTPlants and their seeds have been shown to be a rich source of cystine-stabilized peptides. Recently a new family of plant seed peptides whose sequences are buried within precursors for seed storage vicilins was identified. Members of this Vicilin Buried Peptide (VBP) family are found in distantly related plant species including the monocot date palm, as well as dicotyledonous species like pumpkin and sesame. Genetic evidence for their widespread occurrence indicates that they are of ancient origin. Limited structural studies have been conducted on VBP family members, but two members have been shown to adopt a helical hairpin fold. We here present an extensive characterization of VBPs using solution NMR spectroscopy, to better understand their structural features. Four peptides were produced by solid phase peptide synthesis and shown to adopt a helix-loop-helix hairpin fold, as a result of the I-IV/II-III ladder-like connectivity of their disulfide bonds. Inter-helix interactions, including hydrophobic contacts and salt bridges, are critical for the fold stability and control the angle at which the anti-parallel α-helices interface. Activities reported for VBPs include trypsin inhibitory activity and inhibition of ribosomal function, however their diverse structural features despite a common fold suggest additional bioactivities yet to be revealed are likely.

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Structural Characterization of the PawL-Derived Peptide Family, an Ancient Subfamily of Orbitides

10.1101/2021.07.18.452857 ◽

2021 ◽

Author(s):

Colton D. Payne ◽

Mark F. Fisher ◽

Joshua S. Mylne ◽

K. Johan Rosengren

Keyword(s):

Disulfide Bonds ◽

Solid Phase ◽

Structural Characteristics ◽

Solid Phase Peptide Synthesis ◽

Large Family ◽

Seed Storage ◽

Structural Features ◽

Therapeutic Effects ◽

Evolutionary Origins

Plants are an excellent source of bioactive peptides, often with disulfide bonds and/or a cyclic backbone. While focus has predominantly been directed at disulfide-rich peptides, a large family of small, cyclic but non-disulfide bonded peptides known as the orbitides has been relatively ignored. What research has been conducted has focused on discovering bioactive orbitides, with less attention to structural features. A recently discovered subfamily of the orbitides known as the PawL-Derived Peptides (PLPs) are produced during the maturation of precursors for seed storage albumins. Although their evolutionary origins have been dated, in-depth exploration of the family's structural characteristics and potential bioactivities remains to be conducted. Here we present an extensive characterization of the PLP family. Nine PLPs were produced by solid phase peptide synthesis. Although orbitides can have antimicrobial and therapeutic effects, none of these nine PLPs showed antibacterial or antifungal activity. Their structural features were studied using solution NMR spectroscopy and seven were found to possess regions of backbone order. Ordered regions consist of β-turns, with some PLPs adopting two well-defined β-turns within sequences as short as seven residues, which are largely the result of side chain interactions. Our data highlight that the sequence diversity within this family results in equally diverse shapes.

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Synthesis of NickFects, a New Family of CPPs, by Solid-Phase Peptide Synthesis

Methods in Molecular Biology - Peptide Synthesis ◽

10.1007/978-1-0716-0227-0_16 ◽

2019 ◽

pp. 239-247

Author(s):

Piret Arukuusk ◽

Ülo Langel

Keyword(s):

Peptide Synthesis ◽

Solid Phase ◽

Solid Phase Peptide Synthesis ◽

New Family

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Buckwheat trypsin inhibitor with helical hairpin structure belongs to a new family of plant defence peptides

Biochemical Journal ◽

10.1042/bj20120548 ◽

2012 ◽

Vol 446 (1) ◽

pp. 69-77 ◽

Cited By ~ 37

Author(s):

Peter B. Oparin ◽

Konstantin S. Mineev ◽

Yakov E. Dunaevsky ◽

Alexander S. Arseniev ◽

Mikhail A. Belozersky ◽

...

Keyword(s):

Trypsin Inhibitor ◽

Disulfide Bonds ◽

Plant Defence ◽

Three Dimensional ◽

Structural Similarity ◽

Reversed Phase ◽

Dimensional Structure ◽

Amino Acid Residues ◽

New Family ◽

Helical Hairpin

A new peptide trypsin inhibitor named BWI-2c was obtained from buckwheat (Fagopyrum esculentum) seeds by sequential affinity, ion exchange and reversed-phase chromatography. The peptide was sequenced and found to contain 41 amino acid residues, with four cysteine residues involved in two intramolecular disulfide bonds. Recombinant BWI-2c identical to the natural peptide was produced in Escherichia coli in a form of a cleavable fusion with thioredoxin. The 3D (three-dimensional) structure of the peptide in solution was determined by NMR spectroscopy, revealing two antiparallel α-helices stapled by disulfide bonds. Together with VhTI, a trypsin inhibitor from veronica (Veronica hederifolia), BWI-2c represents a new family of protease inhibitors with an unusual α-helical hairpin fold. The linker sequence between the helices represents the so-called trypsin inhibitory loop responsible for direct binding to the active site of the enzyme that cleaves BWI-2c at the functionally important residue Arg19. The inhibition constant was determined for BWI-2c against trypsin (1.7×10−10 M), and the peptide was tested on other enzymes, including those from various insect digestive systems, revealing high selectivity to trypsin-like proteases. Structural similarity shared by BWI-2c, VhTI and several other plant defence peptides leads to the acknowledgement of a new widespread family of plant peptides termed α-hairpinins.

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Conservation of Amino Acids into Multiple Alignments Involved in Pairwise Interactions in Three-Dimensional Protein Structures

Journal of Bioinformatics and Computational Biology ◽

10.1142/s0219720003000228 ◽

2003 ◽

Vol 01 (03) ◽

pp. 505-520 ◽

Cited By ~ 1

Author(s):

Mounir Errami ◽

Christophe Geourjon ◽

Gilbert Deléage

Keyword(s):

Amino Acids ◽

Disulfide Bonds ◽

Hydrophobic Interactions ◽

Protein Structures ◽

Three Dimensional ◽

Structural Features ◽

Multiple Alignment ◽

Salt Bridges ◽

Multiple Alignments ◽

Multiple Sequences

We present an original strategy, that involves a bioinformatic software structure, in order to perform an exhaustive and objective statistical analysis of three-dimensional structures of proteins. We establish the relationship between multiple sequences alignments and various structural features of proteins. We show that amino acids implied in disulfide bonds, salt bridges and hydrophobic interactions are particularly conserved. Effects of identity, global similarity within alignments, and accessibility of interactions have been studied. Furthermore, we point out that the more variable the sequences within a multiple alignment, the more informative the multiple alignment. The results support multiple alignments usefulness for predictions of structural features.

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Identification, Synthesis, Conformation and Activity of an Insulin-like Peptide from a Sea Anemone

Biomolecules ◽

10.3390/biom11121785 ◽

2021 ◽

Vol 11 (12) ◽

pp. 1785

Author(s):

Michela L. Mitchell ◽

Mohammed Akhter Hossain ◽

Feng Lin ◽

Ernesto L. Pinheiro-Junior ◽

Steve Peigneur ◽

...

Keyword(s):

Growth Factor ◽

Disulfide Bonds ◽

Solid Phase ◽

Solid Phase Peptide Synthesis ◽

Growth Factor Receptors ◽

Sea Anemone ◽

Insulin Like Growth Factor ◽

Functional Studies ◽

The Individual

The role of insulin and insulin-like peptides (ILPs) in vertebrate animals is well studied. Numerous ILPs are also found in invertebrates, although there is uncertainty as to the function and role of many of these peptides. We have identified transcripts with similarity to the insulin family in the tentacle transcriptomes of the sea anemone Oulactis sp. (Actiniaria: Actiniidae). The translated transcripts showed that these insulin-like peptides have highly conserved A- and B-chains among individuals of this species, as well as other Anthozoa. An Oulactis sp. ILP sequence (IlO1_i1) was synthesized using Fmoc solid-phase peptide synthesis of the individual chains, followed by regioselective disulfide bond formation of the intra-A and two interchain disulfide bonds. Bioactivity studies of IlO1_i1 were conducted on human insulin and insulin-like growth factor receptors, and on voltage-gated potassium, sodium, and calcium channels. IlO1_i1 did not bind to the insulin or insulin-like growth factor receptors, but showed weak activity against KV1.2, 1.3, 3.1, and 11.1 (hERG) channels, as well as NaV1.4 channels. Further functional studies are required to determine the role of this peptide in the sea anemone.

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A mesophilic cysteine-less split intein for protein trans-splicing applications under oxidizing conditions

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.1909825116 ◽

2019 ◽

Vol 116 (44) ◽

pp. 22164-22172 ◽

Cited By ~ 13

Author(s):

Maniraj Bhagawati ◽

Tobias M. E. Terhorst ◽

Friederike Füsser ◽

Simon Hoffmann ◽

Tim Pasch ◽

...

Keyword(s):

Disulfide Bonds ◽

Mammalian Cells ◽

Solid Phase ◽

Solid Phase Peptide Synthesis ◽

Reducing Agents ◽

Cell Surface Receptor ◽

Protein Chemistry ◽

Specific Chemical ◽

Ambient Temperatures ◽

Split Intein

Split intein-mediated protein trans-splicing has found extensive applications in chemical biology, protein chemistry, and biotechnology. However, an enduring limitation of all well-established split inteins has been the requirement to carry out the reaction in a reducing environment due to the presence of 1 or 2 catalytic cysteines that need to be in a reduced state for splicing to occur. The concomitant exposure of the fused proteins to reducing agents severely limits the scope of protein trans-splicing by excluding proteins sensitive to reducing conditions, such as those containing critical disulfide bonds. Here we report the discovery, characterization, and engineering of a completely cysteine-less split intein (CL intein) that is capable of efficient trans-splicing at ambient temperatures, without a denaturation step, and in the absence of reducing agents. We demonstrate its utility for the site-specific chemical modification of nanobodies and an antibody Fc fragment by N- and C-terminal trans-splicing with short peptide tags (CysTag) that consist of only a few amino acids and have been prelabeled on a single cysteine using classical cysteine bioconjugation. We also synthesized the short N-terminal fragment of the atypically split CL intein by solid-phase peptide synthesis. Furthermore, using the CL intein in combination with a nanobody–epitope pair as a high-affinity mediator, we showed chemical labeling of the extracellular domain of a cell surface receptor on living mammalian cells with a short CysTag containing a synthetic fluorophore. The CL intein thus greatly expands the scope of applications for protein trans-splicing.

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Total Chemical Synthesis of a Heterodimeric Interchain Bis-Lactam-Linked Peptide: Application to an Analogue of Human Insulin-Like Peptide 3

International Journal of Peptides ◽

10.1155/2013/504260 ◽

2013 ◽

Vol 2013 ◽

pp. 1-8 ◽

Cited By ~ 11

Author(s):

John Karas ◽

Fazel Shabanpoor ◽

Mohammed Akhter Hossain ◽

James Gardiner ◽

Frances Separovic ◽

...

Keyword(s):

Chemical Synthesis ◽

Peptide Synthesis ◽

Disulfide Bonds ◽

Solid Phase ◽

Solid Phase Peptide Synthesis ◽

Protecting Groups ◽

Model Peptide ◽

Design Elements

Nonreducible cystine isosteres represent important peptide design elements in that they can maintain a near-native tertiary conformation of the peptide while simultaneously extending the in vitro and in vivo half-life of the biomolecule. Examples of these cystine mimics include dicarba, diselenide, thioether, triazole, and lactam bridges. Each has unique physicochemical properties that impact upon the resulting peptide conformation. Each also requires specific conditions for its formation via chemical peptide synthesis protocols. While the preparation of peptides containing two lactam bonds within a peptide is technically possible and reported by others, to date there has been no report of the chemical synthesis of a heterodimeric peptide linked by two lactam bonds. To examine the feasibility of such an assembly, judicious use of a complementary combination of amine and acid protecting groups together with nonfragment-based, total stepwise solid phase peptide synthesis led to the successful preparation of an analogue of the model peptide, insulin-like peptide 3 (INSL3), in which both of the interchain disulfide bonds were replaced with a lactam bond. An analogue containing a single disulfide-substituted interchain lactam bond was also prepared. Both INSL3 analogues retained significant cognate RXFP2 receptor binding affinity.

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A Strategy for Thioamide Incorporation During Fmoc Solid-Phase Peptide Synthesis with Robust Stereochemical Integrity

10.26434/chemrxiv.8206247.v1 ◽

2019 ◽

Cited By ~ 1

Author(s):

luis camacho III ◽

Bryan J. Lampkin ◽

Brett VanVeller

Keyword(s):

Amino Acid ◽

Functional Groups ◽

Peptide Synthesis ◽

Solid Phase ◽

Solid Phase Peptide Synthesis ◽

Side Chains ◽

Amino Acid Side Chains ◽

Peptide Elongation

We describe a method to protect the sensitive stereochemistry of the thioamide—in analogy to the protection of the functional groups of amino acid side chains—in order to preserve the thioamide moiety during peptide elongation.<br>

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