Photo-control of bimolecular reactions: reactivity of the long-lived Rhodamine 6G triplet excited state with ˙NO.

Photo-chemistry provides a non-intuitive but very powerful way to probe kinetically limited, sometimes thermodynamically non-favored reactions and, thus, access highly specific products. However, reactivity in the excited state is difficult to characterize directly, due to short lifetimes and challenges in controlling the reaction medium. Among photo-activatable reagents, rhodamine dyes find widespread uses due to a number of favorable properties including their high absorption coefficient. Their readily adaptable synthesis allows development of tailor-made dyes for specific applications. Remarkably, few studies have directly probed the chemical reactivity of their triplet excited state. Here we present a new conceptual approach to examine the specific chemistry of the triplet excited state. We have developed a pump (488 nm) - probe (600 nm) strategy to examine the gas-phase lifetime and reactivity of the triplet cation of Rhodamine 6G (3Rh6G+) in an ion trap mass spectrometer. The confounding effects of solvent, aggregation and formation of other reactive intermediates is thus avoided allowing fundamental reactivity to be explored. In the presence, in the ion trap, of helium seeded with 1% of nitric oxide (˙NO) (∼ 60 ion/˙NO collisions per second), the triplet lifetime is shortened from 1.9 s to 0.7 s. Simultaneously, the reaction products [Rh6G-H]˙+ and [Rh6G-H + NO]+ are observed. Reaction of 3Rh6G+ with ˙NO2 yields [Rh6G-H]˙+, [Rh6G-H + NO2]+ and [Rh6G-2H]+. None of these products are observed for the singlet, 1Rh6G+. DFT calculations suggest a stepwise mechanism only allowed from 3Rh6G+, in which H atom abstraction by ˙NOx (x = 1 or 2) yields [Rh6G-H]˙+ which, then, reacts with another ˙NOx molecule. This illustrates the power of light to initiate specific chemical reactions, and the relevance of gas-phase ion-molecule reaction approaches to understand stepwise reaction mechanism from specific excited states.

Extended disorder at the cell surface: The conformational landscape of the ectodomains of syndecans.

Syndecans are membrane proteoglycans regulating extracellular matrix assembly, cell adhesion and signaling. Their ectodomains can be shed from the cell surface, and act as paracrine and autocrine effectors or as competitors of full-length syndecans. We report the first biophysical characterization of the recombinant ectodomains of the four human syndecans using biophysical techniques, and show that they behave like flexible random-coil intrinsically disordered proteins, and adopt several conformation ensembles in solution. We have characterized their conformational landscapes using native mass spectrometry (MS) and ion-mobility MS, and demonstrated that the syndecan ectodomains explore the majority of their conformational landscape, from minor compact, globular-like, conformations to extended ones. We also report that the ectodomain of syndecan-4, corresponding to a natural isoform, is able to dimerize via a disulfide bond. We have generated a three-dimensional model of the C-terminus of this dimer, which supports the dimerization via a disulfide bond. Furthermore, we have mapped the NXIP adhesion motif of syndecans and their sequences involved in the formation of ternary complexes with integrins and growth factor receptors on the major conformations of their ectodomains, and shown that these sequences are not accessible in all the conformations, suggesting that only some of them are biologically active. Lastly, although the syndecan ectodomains have a far lower number of amino acid residues than their membrane partners, their intrinsic disorder and flexibility allow them to adopt extended conformations, which have roughly the same size as the cell surface receptors (e.g., integrins and growth factor receptors) they bind to.

Isolated Collagen Mimetic Peptide Assemblies Have Stable Triple-Helix Structures.

The origin of the triple-helix structure and high stability of collagen has been debated for many years. As models of the triple helix and building blocks for new biomaterials, collagen mimetic peptide (CMP) assemblies have been deeply studied in the condensed phase. In particular, it was found that hydroxylation of proline, an abundant post-translational modification in collagen, increases its stability. Two main hypotheses emerged to account for this behavior: 1) intra-helix stereoelectronic effects, and 2) the role of water molecules H-bound to hydroxyproline side-chains. However, in condensed-phase investigations, the influence of water cannot be fully removed. Therefore, we employed a combination of tandem ion mobility and mass spectrometries to assess the structure and stability of CMP assemblies in the gas phase. These results show a conservation of the structure and stability properties of triple helix models in the absence of solvent, supporting an important role of stereoelectronic effects. Moreover, evidence that small triple helix assemblies with controlled stoichiometry can be studied in the gas phase is given, which opens new perspectives in the understanding of the first steps of collagen fiber growth.

Visible Multiphoton Dissociation of Chromophore-Tagged Peptides.

The visible photodissociation mechanisms of QSY7-tagged peptides of increasing size have been investigated by coupling a mass spectrometer and an optical parametric oscillator laser beam. The experiments herein consist of energy resolved collision- and laser-induced dissociation measurements on the chromophore-tagged peptides. The results show that fragmentation occurs by similar channels in both activation methods, but that the branching ratios are vastly different. Observation of a size-dependent minimum laser pulse energy required to induce fragmentation, and collisional cooling rates in time resolved experiments show that laser-induced dissociation occurs through the absorption of multiple photons by the chromophore and the subsequent heating through vibrational energy redistribution. The differences in branching ratio between collision- and laser-induced dissociation can then be understood by the highly anisotropic energy distribution following absorption of a photon. Graphical Abstract ᅟ.