Introduction: Structural studies of biomolecules in the gas phase not only reveal their intrinsic protein properties (i.e., those in the absence of solvent) but also provide useful benchmarks for biomolecular modeling. Fluorescence-based techniques like Förster Resonance Energy Transfer (FRET) are among the most popular methods to investigate the structure and dynamics of biomolecules in solution. Analogous explorations in the gas phase have also started to reveal information for the structure elucidation of gaseous biomolecules.[1] However, conventional FRET has its limitations: it can measure distances only in the range of 20 – 80 Å, the size of dyes and linkers renders the distance measurement less accurate, and labeling and purifying a biomolecule with a dye pair is difficult and expensive. Transition metal ion FRET (tmFRET), with a transition metal ion as an acceptor, has emerged as a viable alternative to conventional FRET and solves many of its inherent problems.[2]

Methods: A 16 amino acid helical peptide was labeled with rhodamine 110 fluorophore as a donor at one end and a histidine pair was placed in the helix at a strategic location to bind a transition metal ion as an acceptor. The experiments were performed on a quadrupole ion trap (QIT) mass spectrometer, which was modified to enable gas-phase fluorescence spectroscopy of the trapped ions. This newly modified QIT enables gas-phase fluorescence experiments with a high fluorescence collection efficiency of ~2%. A 10 μM solution of the labeled peptide was electrosprayed in the absence and presence of 40 μM Cu²⁺. Gas-phase fluorescence spectra and fluorescence lifetime decay measurements were separately recorded for the 3+ charge state of the labeled peptide with and without bound Cu²⁺. The fluorescence lifetimes can also be used to estimate distances.

Results: The goal of our study is to perform tmFRET in the gas phase and to use it as a tool to understand the structure of gaseous biomolecular ions. The gas-phase fluorescence spectrum of the donor and solution-phase absorbance spectrum of the acceptor show significant overlap, indicating the possibility of tmFRET. A decrease in gas-phase fluorescence lifetime in the presence of an acceptor further confirms this hypothesis. The fluorescence lifetime of the 3+ charge state of the labeled peptide was found to be 6.0 ns in the absence of an acceptor but decreases to 2.2 ns when complexed with Cu²⁺. This decrease in fluorescence lifetime is caused by FRET between rhodamine 110 and Cu²⁺. From the measured fluorescence lifetimes and estimated critical FRET distance, the distance between the dye and the Cu²⁺ was calculated to be 1.4 nm.

Conclusions: Here we present the first evidence of tmFRET in the gas phase. It does not only add a new tool to the arsenal of gas-phase structural biology but also provides an opportunity for more chemically controlled tmFRET.

[1] Czar, M. F. & Jockusch, R. A., Curr. Opin. Struct. Biol., 2015, 34, 123–134
[2] Taraska, J. W., Puljung, M. C. & Zagotta, W. N., Proc. Natl. Acad. Sci. U. S. A., 2009, 106, 16227–16232.