The use of Forster resonance energy transfer (FRET) as a probe of the structure of biological molecules through fluorescence measurements in solution is well-attested. The transposition of this technique to the gas phase is appealing since it opens the perspective of combining the structural accuracy of FRET with the specificity and selectivity of mass spectrometry (MS). Here, we report FRET results on gasphase polyalanine ions obtained by measuring FRET efficiency through specific photofragmentation rather than fluorescence. The structural sensitivity of the method was tested using commercially available chromophores (QSY 7 and carboxyrhodamine 575) grafted on a series of small, alanine-based peptides of differing sizes. The photofragmentation of these systems was investigated through action spectroscopy, and their conformations were probed using ion mobility spectrometry (IMS) and Monte Carlo minimization (MCM) simulations. We show that specific excitation of the donor chromophore results in the observation of fragments that are specific to the electronic excitation of the acceptor chromophore. This shows that energy transfer took place between the two chromophores and hence that the action-FRET technique can be used as a new and sensitive probe of the structure of gas-phase biomolecules, which opens perspectives as a new tool in structural biology.F orster resonance energy transfer (FRET) is a widely used probe of molecular structure in solution. 1−4 It requires a photon source to electronically excite the so-called "donor chromophore" and a light-harvesting setup to detect either the "donor" or "acceptor" chromophore fluorescence. The occurrence of FRET is then usually evidenced through a decrease in the fluorescence of the donor chromophore (quenching), with the concurrent onset of the fluorescence of the acceptor chromophore or by changes in fluorescence decay times. The interpretation of FRET results relies on the known distance dependence of the effect and on the possibility to graft specific chromophores at relatively well-defined sites on a molecule. FRET is then used to characterize the distance between the chromophores and hence separation between the grafting sites, although extracting exact distances is difficult due to the uncertainty of the exact orientation of the transition dipole moments of the chromophores. This allows the use of FRET to probe intra-or intermolecular distances, especially the change in distance, depending on whether the chromophores are attached to the same or to different molecules.The versatility of FRET makes it a powerful tool to assess the conformation and/or association of molecules. It has been shown that the overall structure of complex molecular edifices can be preserved in the gas phase using soft ionization techniques. 5,6 Therefore, the development of techniques capable of probing FRET in the gas phase is of high interest and could be integrated into a global approach for structure determination of proteins and protein complexes. 7−9 There are few tec...

The gas phase conformations of two amyloid beta mutants are studied by multiple techniques to elucidate the origin of the different aggregation behaviour.

Noncovalent interactions between several protonated amines and an original ditopic receptor, nor‐seco‐cucurbit[10]uril, are investigated by combining mass spectrometry‐based methods and computational chemistry. Electrospray ionization is used to transfer the intact supramolecular assemblies from their acidic solution to the gas phase, provided fine‐tuning of the source parameters is achieved. Ternary complexes, associating two guest molecules and one host cavity, are observed systematically in the mass spectrometry analyses and the quasi‐exclusive occurrence of these 2:1 associations reveals the allosteric nature of the complexation reaction. It is demonstrated that the binary 1:1 complex ions that are detected arise from collision‐induced dissociation processes undergone by the ternary complex ions inside the ion source. Based on ion mobility experiments supported by theoretical calculations, the inclusion nature of the gas‐phase ternary complexes is clearly evidenced independent of the size of the probed guest molecule and the charge state of the complex ions. The allosteric nature of the complexation reactions is dictated by size criteria. This is demonstrated on the basis of mass spectrometry experiments by analyzing solutions containing ligands of different sizes in competition for inclusion within the guest cavity. Computational chemistry is also used to characterize the three‐dimensional structures of the complexes.