Editorial pubs.acs.org/ac
Coming of Age: Gas-Phase Structural Information on Biomolecules by FRET
F
örster resonant energy transfer (FRET) is a strongly distance sensitive method and can be used as a “spectroscopic ruler” to obtain distance constraints within a molecule and low-resolution structural information, as shown in the landmark study by Stryer more than 60 years ago for poly-Pro of different lengths.1 In FRET experiments, a donor fluorophore conjugated to a part of the (bio)molecule absorbs photons of a specific wavelength. If the distance to an acceptor chromophore, which is conjugated to another part of the biomolecule is small enough, radiationless (“Förster”) energy transfer occurs, and significantly red-shifted, “sensitized” emission from the acceptor fluorophore is observed. There is significant interest to carry out FRET measurement in the gas phase, 2,3 fueled by developments in the mass spectrometry community to carry out “native” electrospray ionization to obtain gas-phase biomolecular ions with a conformation that is presumably close to the native one. Whether this is fact or fiction is an unsolved question. Unfortunately, canonical FRET is difficult to carry out in the gas phase, because the number of trapped ions is limited, optical access to the ion cloud is difficult, and the optical properties of fluorophores in the gas phase differ from those in solution (see Figure 1).
Figure 2. TOC graphic from ref 4 illustrating the principle of “Action FRET”. Upon excitation of the donor, and depending on the distance between the chromophores, FRET occurs more or less efficiently (lower right). In contrast to optically detected FRET, specific fragmentation is observed in the vicinity of the acceptor moiety, leading to the peaks at m/z = 360, 465, and 531 (lower left), which are used as reporter ions for the FRET efficiency. Reprinted from ref 5. Copyright 2014 American Chemical Society.
In a new paper in this issue,5 the group of Jockusch in Toronto used an experimental setup that combines trapped ion mass spectrometry and laser-induced fluorescence (i.e., optically detected FRET) to probe the structure of the protein GB1 as a function of charge state. Steady-state fluorescence emission spectra and time-resolved donor fluorescence measurements of mass-selected GB1 gave consistent results, both showing a marked decrease in the FRET efficiency with increasing number of charges on the gaseous protein, which suggests a Coulombically driven unfolding and expansion of the structure. This lies in stark contrast to the pH stability of GB1 in solution. Comparison with solution-phase single-molecule FRET measurements show lower FRET efficiency for all charge states of the gaseous protein examined, indicating that the ensemble of conformations present in the gas phase is, on average, more expanded than the native form. These results represent the first canonical FRET measurements on a mass-selected protein in the gas phase. Both studies are of great fundamental as well as methodological interest, and it is both exciting and gratifying to see these published in Analytical Chemistry. This research illustrates the utility of FRET for obtaining new kinds of structural information for large, desolvated biomolecules in the gas phase, opening perspectives as a new tool in the emerging field of “gas-phase structural biology”.
Figure 1. TOC graphic from ref 5 illustrating the principle of FRET and how the multiply charged gas-phase ions produced in electrospray ionization may cause unfolding of the protein. Reprinted from ref 5. Copyright 2015 American Chemical Society.
Two landmark papers have appeared in Analytical Chemistry on gas-phase FRET within less than a year, which motivated me to write this guest editorial. The group of Dugourd in Lyon developed “Action FRET” where specific dissociation of the acceptor chromophore (a quencher, in this case) and parts of the molecule close to the acceptor instead of photoemission from an acceptor is employed to report on the spatial proximity of the donor and acceptor chromophores.4 Action FRET circumvents many of the difficulties of optically detected FRET and is more sensitive: ionic products rather than light are detected, which avoids many problems inherent in optically detected FRET of stored gas-phase ions. Daly et al. tested the structural sensitivity of the method using commercially available chromophores grafted on a series of small, alanine-based peptides of differing sizes (see Figure 2). © 2015 American Chemical Society
Published: July 9, 2015 7497
DOI: 10.1021/acs.analchem.5b02456 Anal. Chem. 2015, 87, 7497−7498
Analytical Chemistry
Editorial
Renato Zenobi, Ph.D., Associate Editor Analytical Chemistry
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Department of Chemistry and Applied Biosciences, Eidgenössische Technische Hochschule (ETH) Zürich, CH-8093 Zürich, Switzerland
AUTHOR INFORMATION
Notes
Views expressed in this editorial are those of the author and not necessarily the views of the ACS. E-mail:
[email protected].
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REFERENCES
(1) Stryer, L. Annu. Rev. Biochem. 1978, 47, 819. (2) Dashtiev, M.; Azoz, V.; Frankevich, V.; Zenobi, R. J. Am. Soc. Mass Spectrom. 2005, 16, 1481. (3) Danell, A. S.; Parks, J. H. Int. J. Mass Spectrom. 2003, 229, 35−45. (4) Daly, S.; et al. Anal. Chem. 2014, 86, 8798−8804. (5) Czar, M. F.; et al. Anal. Chem. 2015, DOI: 10.1021/ acs.analchem.5b01591.
7498
DOI: 10.1021/acs.analchem.5b02456 Anal. Chem. 2015, 87, 7497−7498
