6.2
Fluorescence as a Probe of Structural Changes
of Biomolecular Ions
Maybe the largest potential of gas-phase emission spectroscopy is to use it to probe
DNA and protein conformations and dynamics based on Fo ¨rster Resonance Energy
Transfer (FRET) [1] or photoinduced electron transfer where the biomolecule is
labelled with one or more fluorescent probes. Indeed, changes in fluorescence
intensity can be correlated with changes in the average conformation of the
molecule as the rate of energy transfer between a donor–acceptor pair strongly
depends on their separation, to the inverse power of six. The absorption band of the
donor is normally to the blue of that of the acceptor and likewise for the emission
bands, but such that the donor emission band overlaps with the acceptor absorption
band. The photo-excited donor transfers its excitation energy to the acceptor as a
result of long-range dipole–dipole interactions; no photon emission and photon
absorption is involved in the RET process! As the FRET method is distancesensitive, it can be used as a “molecular ruler”. The distance between the donor
and acceptor at which the energy transfer efficiency is half of its maximum is
denoted the Fo ¨rster distance, R 0 , and is typically 20–60 A ˚ , which renders this
method optimum for the study of biomolecular structures. In summary, if the
donor is selectively photoexcited (possible as the absorption spectra of the donor
and acceptor are different) and the acceptor is far away, the emission will be solely
from the donor. If the acceptor is close to the photo-excited donor, RET will take
place to a certain extent, and there will be both donor and acceptor emission. At
optimum distance between the two, the donor is fully quenched, and only acceptor
emission is seen (100 % RET efficiency).
Pioneering work on gas-phase biomolecular ions relating to FRET has been done
by Parks, Zenobi, Jockusch and their co-workers [15–17]. Some of their work will
be summarised in the following.
Danell and Parks [15] monitored the separation of double-stranded oligonucleotide anions (i.e., duplex melting) based on FRET using two dyes (the BODIPYTMR donor and the BODIPY-TR acceptor), one on each single strand and attached
to the ends so that they are in close proximity for the intact duplex. The model
duplex was composed of two complementary 14-mer strands that formed seven AT
Watson–Crick base pairs at one end and seven GC base pairs at the other end (A ¼
adenine, T ¼ thymine, G ¼ guanine, C ¼ cytosine). The dyes were attached to the
AT end as melting expectedly occurs here; cf., only two hydrogen bonds between A
and T versus three between G and C. The donor fluorescence as well as dissociation
was followed as a function of temperature. If the donor separates from the acceptor,
its fluorescence increases as it does not transfer its excitation energy to the acceptor;
in contrast acceptor fluorescence will go down (this light was, however, not
detected in the experiment). The results showed clear evidence for an intermediate
state (partly “unzipped” duplex at the AT end) preceding dissociation (Fig. 6.1).
While dissociation was easily established by mass spectrometry, the intermediate
state has the same m/z as the initial duplex, and its formation could not be inferred
6 Fluorescence from Gas-Phase Biomolecular Ions
107
Fluorescence as a Probe of Structural Changes
of Biomolecular Ions
Maybe the largest potential of gas-phase emission spectroscopy is to use it to probe
DNA and protein conformations and dynamics based on Fo ¨rster Resonance Energy
Transfer (FRET) [1] or photoinduced electron transfer where the biomolecule is
labelled with one or more fluorescent probes. Indeed, changes in fluorescence
intensity can be correlated with changes in the average conformation of the
molecule as the rate of energy transfer between a donor–acceptor pair strongly
depends on their separation, to the inverse power of six. The absorption band of the
donor is normally to the blue of that of the acceptor and likewise for the emission
bands, but such that the donor emission band overlaps with the acceptor absorption
band. The photo-excited donor transfers its excitation energy to the acceptor as a
result of long-range dipole–dipole interactions; no photon emission and photon
absorption is involved in the RET process! As the FRET method is distancesensitive, it can be used as a “molecular ruler”. The distance between the donor
and acceptor at which the energy transfer efficiency is half of its maximum is
denoted the Fo ¨rster distance, R 0 , and is typically 20–60 A ˚ , which renders this
method optimum for the study of biomolecular structures. In summary, if the
donor is selectively photoexcited (possible as the absorption spectra of the donor
and acceptor are different) and the acceptor is far away, the emission will be solely
from the donor. If the acceptor is close to the photo-excited donor, RET will take
place to a certain extent, and there will be both donor and acceptor emission. At
optimum distance between the two, the donor is fully quenched, and only acceptor
emission is seen (100 % RET efficiency).
Pioneering work on gas-phase biomolecular ions relating to FRET has been done
by Parks, Zenobi, Jockusch and their co-workers [15–17]. Some of their work will
be summarised in the following.
Danell and Parks [15] monitored the separation of double-stranded oligonucleotide anions (i.e., duplex melting) based on FRET using two dyes (the BODIPYTMR donor and the BODIPY-TR acceptor), one on each single strand and attached
to the ends so that they are in close proximity for the intact duplex. The model
duplex was composed of two complementary 14-mer strands that formed seven AT
Watson–Crick base pairs at one end and seven GC base pairs at the other end (A ¼
adenine, T ¼ thymine, G ¼ guanine, C ¼ cytosine). The dyes were attached to the
AT end as melting expectedly occurs here; cf., only two hydrogen bonds between A
and T versus three between G and C. The donor fluorescence as well as dissociation
was followed as a function of temperature. If the donor separates from the acceptor,
its fluorescence increases as it does not transfer its excitation energy to the acceptor;
in contrast acceptor fluorescence will go down (this light was, however, not
detected in the experiment). The results showed clear evidence for an intermediate
state (partly “unzipped” duplex at the AT end) preceding dissociation (Fig. 6.1).
While dissociation was easily established by mass spectrometry, the intermediate
state has the same m/z as the initial duplex, and its formation could not be inferred
6 Fluorescence from Gas-Phase Biomolecular Ions
107