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Equipping Coiled-Coil Peptide Dimers With Furan Warheads Reveals Novel Cross-Link Partners.

ABSTRACT: Using a coiled-coil peptide dimer as a model system to explore furan reactivity, we describe novel cross-link partners of furan warheads for site-specific cross-linking. We demonstrate that replacement of weak interhelical ionic contacts with a furan moiety and its potential cross-link partner affords covalently connected coiled-coil motifs upon furan activation. We describe for the first time the reaction of the activated furan warhead with cysteine and tyrosine, besides the previously reported lysine, thus enhancing the versatility of the furan cross-link methodology by the possibility to target different amino acids. The present in vitro validation of "furan-armed" α-helices provides further grounds for exploiting furan technology in the development of furan-modified ligands/proteins to target proteins in a covalent way through various amino acid side chains.

SUBMITTER: Miret-Casals L

PROVIDER: S-EPMC8888431 | biostudies-literature | 2021

REPOSITORIES: biostudies-literature

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Publications

Equipping Coiled-Coil Peptide Dimers With Furan Warheads Reveals Novel Cross-Link Partners.

Miret-Casals Laia L Van De Putte Sander S Aerssens Dorien D Diharce Julien J Bonnet Pascal P Madder Annemieke A

Frontiers in chemistry 20220216

Using a coiled-coil peptide dimer as a model system to explore furan reactivity, we describe novel cross-link partners of furan warheads for site-specific cross-linking. We demonstrate that replacement of weak interhelical ionic contacts with a furan moiety and its potential cross-link partner affords covalently connected coiled-coil motifs upon furan activation. We describe for the first time the reaction of the activated furan warhead with cysteine and tyrosine, besides the previously reported ...[more]

PMID: 35252125

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Project description:The alpha-helical coiled coil is a structurally simple protein oligomerization or interaction motif consisting of two or more alpha helices twisted into a supercoiled bundle. Coiled coils can differ in their stoichiometry, helix orientation, and axial alignment. Because of the near degeneracy of many of these variants, coiled coils pose a challenge to fold recognition methods for structure prediction. Whereas distinctions between some protein folds can be discriminated on the basis of hydrophobic/polar patterning or secondary structure propensities, the sequence differences that encode important details of coiled-coil structure can be subtle. This is emblematic of a larger problem in the field of protein structure and interaction prediction: that of establishing specificity between closely similar structures. We tested the behavior of different computational models on the problem of recognizing the correct orientation--parallel vs. antiparallel--of pairs of alpha helices that can form a dimeric coiled coil. For each of 131 examples of known structure, we constructed a large number of both parallel and antiparallel structural models and used these to assess the ability of five energy functions to recognize the correct fold. We also developed and tested three sequence-based approaches that make use of varying degrees of implicit structural information. The best structural methods performed similarly to the best sequence methods, correctly categorizing approximately 81% of dimers. Steric compatibility with the fold was important for some coiled coils we investigated. For many examples, the correct orientation was determined by smaller energy differences between parallel and antiparallel structures distributed over many residues and energy components. Prediction methods that used structure but incorporated varying approximations and assumptions showed quite different behaviors when used to investigate energetic contributions to orientation preference. Sequence based methods were sensitive to the choice of residue-pair interactions scored.

Project description:Owing to their synthetic accessibility and protein-mimetic features, peptides represent an attractive biomolecular building block for the fabrication of artificial biomimetic materials with emergent properties and functions. Here, we expand the peptide building block design space through unveiling the design, synthesis, and characterization of novel, multivalent peptide macrocycles (96mers), termed coiled coil peptide tiles (CCPTs). CCPTs comprise multiple orthogonal coiled coil peptide domains that are separated by flexible linkers. The constraints, imposed by cyclization, confer CCPTs with the ability to direct programmable, multidirectional interactions between coiled coil-forming "edge" domains of CCPTs and their free peptide binding partners. These fully synthetic constructs are assembled using a convergent synthetic strategy via a combination of native chemical ligation and Sortase A-mediated cyclization. Circular dichroism (CD) studies reveal the increased helical stability associated with cyclization and subsequent coiled coil formation along the CCPT edges. Size-exclusion chromatography (SEC), analytical high-performance liquid chromatography (HPLC), and fluorescence quenching assays provide a comprehensive biophysical characterization of various assembled CCPT complexes and confirm the orthogonal colocalization between coiled coil domains within CCPTs and their designed on-target free peptide partners. Lastly, we employ molecular dynamics (MD) simulations, which provide molecular-level insights into experimental results, as a supporting method for understanding the structural dynamics of CCPTs and their complexes. MD analysis of the simulated CCPT architectures reveals the rigidification and expansion of CCPTs upon complexation, i.e., coiled coil formation with their designed binding partners, and provides insights for guiding the designs of future generations of CCPTs. The addition of CCPTs into the repertoire of coiled coil-based building blocks has the potential for expanding the coiled coil assembly landscape by unlocking new topologies having designable intermolecular interfaces.

Dataset Information

Equipping Coiled-Coil Peptide Dimers With Furan Warheads Reveals Novel Cross-Link Partners.

Publications

Equipping Coiled-Coil Peptide Dimers With Furan Warheads Reveals Novel Cross-Link Partners.

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