V.M. Trusova1, G.P.
Gorbenko1, T. Deligeorgiev2, A. Vasilev2,
1V.N.
2Department of Applied Organic Chemistry, Faculty of Chemistry,
Novel DONOR-ACCEPTOR
PAIR FOR ENERGY TRANSFER STUDIES
Fluorescence resonance energy transfer
(FRET) is an ultra-powerful physical technique that found numeral applications in a wide
variety of scientific and technological areas. Due to explicit dependence of
FRET efficiency on the distance between fluorophores acting as
energy donor and acceptor, this method proved to be extremely useful for determination
of the molecular proximity within the nanometer scale. The
crucial point in successful application of FRET in structural characterization
of macromolecular assemblies is the appropriate choice of fluorophores.
Ideally, the donor-acceptor (D-A) pair must fulfil the following
requirements:
·
high Förster
overlap integral,
·
high fluorescence quantum yield,
·
spectral
gap between the absorbance of donor and acceptor to avoid any residual
excitation of acceptor molecule,
·
high
photostability,
·
high
affinity to biological macromolecules,
·
minimal
interference of donor/acceptor absorption/emission spectra with those of biological
molecules.
Despite the broad selection of
available fluorophores, D-A pairs that fully meet the above challenges have to
be found. In the present paper we have evaluated the potential of novel D-A
pair represented by 4-p-(dimethylaminostyryl)-1-dodecylpyridinium (DSP-12) as donor and squaraine derivative SQ-1 as
energy acceptor (Fig. 1) which are expected to satisfy the above requirements.
The applicability of these fluorophores for FRET studies has been tested on
lipid vesicles, composed of zwitterionic lipid phosphatidylcholine (PC) and
sterol cholesterol (Chol) in molar ratio 19:1.
DSP-12
and SQ-1 belong to the big family of long-wavelength fluorescent probes. Their
emission and absorption is in so-called optical window where biological
macromolecules are spectroscopically-inactive. Being associated in the membrane,
these dyes reside at the lipid-water interface with the long alkyl chains
buried in the bilayer hydrophobic core. The pair DSP-12–SQ-1 is well-suited for
FRET studies due to several reasons. First, emission spectrum of DSP-12 significantly
overlaps with absorbance spectrum of SQ-1 (Fig. 1). This results in very high
value of Förster radius (ca. 6
nm). Second, DSP-12 absorbs in the wavelength region where SQ-1 absorption is
negligible. Third, excitation band of DSP-12 is sufficiently separated from
that of SQ-1 to allow selective donor excitation. Furthermore, the observations
that i) excitation spectra of SQ-1 is featured by well-defined DSP-12
absorbance band, and ii) increase of SQ-1 concentration resulted in the quenching
of DSP-12 fluorescence and appearance of SQ-1 emission peak in the fluorescence
spectra of DSP-12 (Fig. 2) strongly suggest that these dyes represent an ideal
donor-acceptor pair.
The next step of our study was focused
on quantitative characterization of FRET between DSP-12 and SQ-1. The results
of energy transfer experiments were analyzed in terms of the model proposed by Fung
and Stryer for two-dimensional systems. Assuming that donor and acceptor are
randomly distributed in different planes separated by a distance 
, the efficiency of energy transfer is defined as:
, 
,
where
l=t/td, td is
the lifetime of excited donor in the absence of acceptor, Ro is the Förster radius, 
is the surface acceptor concentration. However,
no satisfactory data fit was achieved in terms of this model. As shown in Fig.
3, experimental points lie below the theoretical curve. Analysis of the
possible reasons for this discrepancy allowed us to conclude that i) donor and
acceptor are located in one plane, and ii) fluorophore mobility is restricted
and nonisotropic.
Overall, the present study provides
indisputable proofs for the applicability of DSP-12 and SQ-1 as donor-acceptor
pair in a variety of biomedical studies.