Optimal Dynamic Discrimination in Tryptophan-Containing Dipeptides
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Optimal Dynamic Discrimination in Tryptophan-Containing Dipeptides
S. Afonina 0
O. Nenadl 0
A. Rondi 0
D. Kiselev 0
J. Extermann 0
L. Bonacina 0
J.-P. Wolf 0
0 GAP - Biophotonics, University of Geneva , Geneva , Switzerland
Optimal Dynamic Discrimination based on the phase-shaping of deep ultraviolet femtosecond pulses was applied to selectively modulate the time-resolved fluorescence depletion of pairs of tryptophan-containing dipeptides. Our results indicate that phase-sensitive excitation allows their differential identification, beyond the limits of linear and time-resolved spectroscopy.
Introduction
The essential amino acid tryptophan (trp) and its dipeptide counterparts alanine-
(alatrp), glycine- (gly-trp), and leucine-tryptophan (leu-trp) are ubiquitous molecules of high
biological significance. In particular, trp is the main probe for UV-vis spectroscopy, thanks to
its high fluorescence quantum yield and large Stokes shift. The high sensitivity of trp to the
environment is well known, and, in this respect, trp bound to a single amino-acid by a peptide
bond represents a perfect workbench for research.
The spectroscopic properties of all the molecules under scrutiny are dominated by those of
trp, and, in fact, they present identical absorption and strongly overlapping florescence
spectra, making their discrimination by linear spectroscopy nearly impossible (Fig. 1a).
Interestingly, even time-resolved approaches (i.e. fluorescence depletion), which proved
successful in the past for discriminating trp-containing samples [1], are not selective enough in
the present case. On the other hand, our group has demonstrated how, within the framework
of Optimal Dynamic Discrimination (ODD) [2], a careful manipulation of pulse spectral-phase
can drive the selective excitation of the low-frequency molecular modes in spectroscopycally
identical biochromophores, eventually leading to their fluorescence-based identification in a
mixture [2,3]. Moreover this method has shown high discrimination power in the fluorescence
depletion experiment for two essential aromatic amino acids: tryptophan and tyrosine. By use
of acousto-optic programmable dispersive filter (AOPDF), commercially known as Dazzler
(Fastlite), fluorescence modulation was achieved with a contrast of 35% [4].
In this contribution, we demonstrate that, by employing a new dedicated reflective shaper
[5], the ODD approach can be readily extended from the UV-visible to the deep UV spectral
region, accessing the absorption bands of amino-acids, peptides, and possibly DNA.
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Results and Discussion
As one can see from Fig. 1, the fluorescence depletion transients measured on trp and the three
dipeptides under study are indistinguishable when close-to-Fourier-Transform pulses are employed
for excitation (266 nm) and depletion (800 nm). To counteract this lack of selectivity, we ran a series
of feedback driven optimizations aimed at enhancing the difference in the fluorescence depletion
response yielded by pairs of molecules interrogated simultaneously. Thus the goal was to maximize
or minimize the quantity of depletion value with respect to the reference fluorescence depletion with
unshaped pulse.
Example of successful optimization experiment for trp and ala-trp one can see on the fig. 2a-c.
More specifically, the spectral phase of the UV pulse was modified according to the evolution of a
multi-objective genetic algorithm [6]. Depletion ratio was obtained from measurement sets (each set
is the average of 1000 measurements) for generating statistically relevant figures.
For several molecule pairs (trp vs ala-trp, trp vs cyclo(-gly-trp), cyclo(-gly-trp) vs ala-trp) the
discrimination capability of the approach is the order of 20%, which is remarkably high considering
the modest bandwidth available at 266 nm (3 nm FWHM). This restricted parameter space does not
allow, in opposite, to discriminate other dipeptides like ala-trp and cyclo(-leu-trp).
Conclusions
We showed the phase-sensitive modulation of the fluorescence depletion of trp and trp-containing
dipeptides with the aim of fluorescence-based identification of otherwise spectrally indistinguishable
molecules. This work demonstrates that ODD can be extended to the manipulation of optical
response of deep UV absorbing biochromophor like proteins and even DNA.
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