Covalent Cross-Linking within Supramolecular Peptide Structures

Abstract

β-Sheet peptide nanostructures (e.g., amyloid fibrils) are recognized as important entities in biological systems and as functional materials in their own right. Their unique physical properties and architectural complexity, however, present a challenge for structure determination at atomic resolution. Covalent cross-linking and mass spectrometry are appealing methods for this endeavor because, potentially, a large amount of information can be extracted from a small sample in a single experiment. Previously, we described preliminary studies on the use of a photoreactive diazirine-containing amino acid to cross-link peptide monomers in nanostructures, together with the integrated separation and analysis of the products using ion mobility spectrometry coupled to conventional mass spectrometry. Here, a pH-switchable system (Aβ(16-22), a sequence from the amyloid-β peptide) was used to examine cross-linking chemistry in morphologically distinct supramolecular structures containing, or entirely composed of, diazirine-functionalized peptides. We examine the relationship between cross-linker chemistry, covalent cross-links (identified using chemical derivatization and tandem mass spectrometry), and noncovalent structure, and report differences in the site of cross-linking that can only be explained by supramolecular templating. The results demonstrate the applicability of the approach for obtaining structural restraints in ordered supramolecular assemblies, provided that a considered evaluation of the cross-linked products is undertaken.

Analysis of Amyloid Nanostructures Using Photo-cross-linking: In Situ Comparison of Three Widely Used Photo-cross-linkers

“…Irradiation of Aβ 16–22 -TFMD 20 at 365 nm generated a distribution of adducts as described previously. 27 Presently, it was noteworthy that a significant quantity of the diazirine remained at 5 min, but that this had been consumed by 60 min. The dominant photolysis product was assigned as a hexafluoroisopropyl ether ( m / z 1142; change in mass, Δ m , = +140), formed via insertion of singlet carbene and/or solvolysis of photoisomerised Aβ 16–22 -TFMD 20 .…”

“…27 Molecularly dissolved peptides were irradiated (254 or 365 nm) for varying amounts of time (typically 0, 5, or 60 min) and analyzed using liquid chromatography (LC) interfaced to an ion trap mass spectrometer. The choice of wavelength was guided by experiment (see UV–vis spectra in Supplementary Figures ESI 5 and ESI 6) and, where necessary, by relevant information from the literature.…”

“…We previously identified the HFIP adduct as resulting from isomerization of TFMD to the diazoisomer which in the presence of mildly acidic HFIP gives a reactive carbocation that is quenched by HFIP. 27 Note that m / z 984 had not appeared when HPLC-purified Aβ 16–22 -TFMD 20 was photolyzed in HFIP ( i.e. , no intermolecular cross-links formed in the absence of supramolecular structure).…”

Enzyme Modification and Oxidative Cross‐linking Using Carboxylate‐, Phenol‐ and Catechol‐Conjugated 1,8‐Naphthalimides

“…Light activation of tris(2,2 0 -bipyridyl) ruthenium(II) complexes, in the presence of a sacrificial oxidant, initiate protein-protein crosslinks via the one-electron oxidation of the amino acid side chain (1,5,6). This technique has been used to study amyloid formations over time that result in neurological disorders like Parkinson's disease (7)(8)(9)(10)(11)(12). The method has also been applied to wound sealing in clinical applications to cross-link tissue after injury (13)(14)(15).…”

SIM-XL: A powerful and user-friendly tool for peptide cross-linking analysis

“…Ultimately, these distance restraints enable a variety of structural information to be obtained, unraveling important information for understanding protein folding, complex topology and interaction regions [1,2].…”