ac7b00152_si_001.pdf (699.75 kB)
Action-Self Quenching: Dimer-Induced Fluorescence Quenching of Chromophores as a Probe for Biomolecular Structure
journal contribution
posted on 2017-03-29, 00:00 authored by Steven Daly, Chang Min Choi, Fabien Chirot, Luke MacAleese, Rodolphe Antoine, Philippe DugourdTo
obtain a more detailed understanding of how structure influences
the function and interaction of biomolecules, it is important to develop
structure sensitive techniques to probe these relationships. Alongside in vivo and in vitro techniques, it is
instructive to consider in vacuo methodologies: for
example native mass spectrometry, ion mobility mass spectrometry,
and FRET. Here, we propose a novel technique for probing biomolecular
structure based on the changes in photophysics of a chromophore upon
dimer formation. Comparison of solution and gas phase measurements
on a doubly tagged tripeptide shows that dimer-induced fluorescence
quenching is accompanied by an increase in photofragmentation yield.
The 12–28 fragment of amyloid beta was used to show that as
the charge state was increasedpreviously shown to cause a
conformational change from compact random coil to extended helical
structurethe disappearance of a band at 495 nm could be correlated
with the level of self-quenching. The presence of features in the
action spectrum of the +3 charge state of both quenched and unquenched
chromophores allowed inference of multiple conformations. Single wavelength
measurements on doubly tagged ubiquitin cations were performed to
show that the technique is feasible on a small protein. These results
demonstrate that self-quenching is a sensitive and fast gas-phase
probe of biomolecular structure that can be directly linked to solution
phase measurements. Further, it is capable of probing very small changes
in conformation, making it complementary to FRET based techniques,
which are insensitive at very short chromophore separations.