contain only a donor or an acceptor display emission spectra similar to those for the
isolated dyes except for small redshifts (compare with Fig. 6.4). However, importantly, photoexcitation of the donor in the DA peptide leads to emission from both
donor and acceptor, which implies that RET has occurred. In line with this, fluorescence lifetimes are longer for D-only peptides and A in DA peptides (photoexcitation
of A) than those for D in DA peptides (photoexcitation of D, here the fluorescence
lifetime refers to the lifetime of the electronically excited state of D) (see Fig. 6.6),
again demonstrating that quenching of the photoexcited donor takes place in the
presence of an acceptor. There are two lifetimes for the DA peptides, which were
associated with two distinct conformational families of the peptide. The authors
found that the n ¼ 8 peptide, where the distance between the two dyes is shortest,
showed almost no fluorescence from the donor independent of the charge state being
2+ or 3+. Hence energy transfer is nearly complete. For n ¼ 14 the FRET efficiency
was found to decrease with an increase in charge state, which is in agreement with the
peptides adopting more extended structures due to the higher Coulomb repulsion.
Surprisingly, the n ¼ 20 peptides with 4+ charge state displayed similar extents of
RET as the n ¼ 14 peptides, which indicates similar dye–dye distances. For the 5+
charge state peptide virtually complete energy transfer occurred suggesting a hair-pin
structure in which the two ends are close together. This work again very nicely shows
the usefulness of FRET to study conformations of biomolecular ions in vacuo.
An interesting question up for much debate is whether macromolecular ions
preserve their solution-phase structures when transferred to the gas phase no matter
how gently this is done. To address this, Parks and co-workers [24] measured the
fluorescence from cytochrome c protein ions within electrospray droplets, that is,
non-isolated ions. There a Trp residue is the light emitter and heme the quencher via
resonance energy transfer at close proximity between the two. High fluorescence is
associated with protein unfolding or refolding of the protein at large alcohol
concentrations. The work revealed significant differences between the protein
conformation in the plume and in a similar solution with various methanol-towater ratios. Care must therefore be taken in establishing the gas-phase structures.
6.3
Light Emission from Fireflies: Emission from the Bare
Oxyluciferin Anion?
In fireflies the oxyluciferin anion is formed in an electronically excited state within
the luciferase enzyme [25]. The precursor is D-luciferin and the chemical reactions
catalysed by the enzyme involve the energy-rich adenosine triphosphate (ATP),
Mg
2+ , and molecular oxygen. The anion can return to the ground state by light
emission (bioluminescence), which occurs with a high efficiency [26]. Interestingly, different species emit light with different wavelengths despite the fact that
they all possess the same luminophore. One explanation is based on various
microenvironments of the oxyluciferin within the protein pocket [27], e.g., single
water molecules or charged amino acid residues. To disentangle the intrinsic
6 Fluorescence from Gas-Phase Biomolecular Ions
111
isolated dyes except for small redshifts (compare with Fig. 6.4). However, importantly, photoexcitation of the donor in the DA peptide leads to emission from both
donor and acceptor, which implies that RET has occurred. In line with this, fluorescence lifetimes are longer for D-only peptides and A in DA peptides (photoexcitation
of A) than those for D in DA peptides (photoexcitation of D, here the fluorescence
lifetime refers to the lifetime of the electronically excited state of D) (see Fig. 6.6),
again demonstrating that quenching of the photoexcited donor takes place in the
presence of an acceptor. There are two lifetimes for the DA peptides, which were
associated with two distinct conformational families of the peptide. The authors
found that the n ¼ 8 peptide, where the distance between the two dyes is shortest,
showed almost no fluorescence from the donor independent of the charge state being
2+ or 3+. Hence energy transfer is nearly complete. For n ¼ 14 the FRET efficiency
was found to decrease with an increase in charge state, which is in agreement with the
peptides adopting more extended structures due to the higher Coulomb repulsion.
Surprisingly, the n ¼ 20 peptides with 4+ charge state displayed similar extents of
RET as the n ¼ 14 peptides, which indicates similar dye–dye distances. For the 5+
charge state peptide virtually complete energy transfer occurred suggesting a hair-pin
structure in which the two ends are close together. This work again very nicely shows
the usefulness of FRET to study conformations of biomolecular ions in vacuo.
An interesting question up for much debate is whether macromolecular ions
preserve their solution-phase structures when transferred to the gas phase no matter
how gently this is done. To address this, Parks and co-workers [24] measured the
fluorescence from cytochrome c protein ions within electrospray droplets, that is,
non-isolated ions. There a Trp residue is the light emitter and heme the quencher via
resonance energy transfer at close proximity between the two. High fluorescence is
associated with protein unfolding or refolding of the protein at large alcohol
concentrations. The work revealed significant differences between the protein
conformation in the plume and in a similar solution with various methanol-towater ratios. Care must therefore be taken in establishing the gas-phase structures.
6.3
Light Emission from Fireflies: Emission from the Bare
Oxyluciferin Anion?
In fireflies the oxyluciferin anion is formed in an electronically excited state within
the luciferase enzyme [25]. The precursor is D-luciferin and the chemical reactions
catalysed by the enzyme involve the energy-rich adenosine triphosphate (ATP),
Mg
2+ , and molecular oxygen. The anion can return to the ground state by light
emission (bioluminescence), which occurs with a high efficiency [26]. Interestingly, different species emit light with different wavelengths despite the fact that
they all possess the same luminophore. One explanation is based on various
microenvironments of the oxyluciferin within the protein pocket [27], e.g., single
water molecules or charged amino acid residues. To disentangle the intrinsic
6 Fluorescence from Gas-Phase Biomolecular Ions
111