XIII European Conference on the Spectroscopy of ... - ecsbm 2009

Palermo, August 28 – September 2, 2009 13 th ECSBM −−−− Book of Abstracts
Fluorescence and Infrared Cross-Correlation Spectroscopy:
A new tool in analysing protein conformational coupling
S. MADATHIL 1 , U. ALEXIEV 2 AND K. FAHMY1 1
1. Div. of Biophysics, Institute of Radiochemistry, Forschungszentrum
Dresden-Rossendorf, PF 510119, D-01314 Dresden, Germany
2. Dept. of Physics, Freie Universität Berlin, Arnimallee 14, D-14195 Berlin, Germany
The functional regulation of proteins is based on structural changes that are initiated at a
ligand-binding site and are transmitted to a "distant" site where biological activity is altered.
Understanding the molecular mechanisms of long range coupling is crucial for many systems
ranging from energy conversion and catalysis to pharmacoligical interference with receptors.
We have developed a generalized multidimensional spectoscopic approach to investigate long
range coupling in proteins by integrating simultaneously recorded fluorescence emission and
infrared absorption from a protein sample undergoing conformational transitions in response
to an external perturbation. Using fluorescence-coupled attenuated total relfectance (ATR)
Fourier-transform infrared (FTIR) difference spectroscopy, coupling of the cytosolic
conformation of the retinal protein rhodopsin to internal carboxyl_H-bonds has thus been
identified [1]. Here we have used the fluorescence-labeled bacteriorhodopsin mutant D96A to
show how the closure of the cytosolic half channel of this proton pump is coupled to the
protonation state of the retinal Schiff base and its counter ion Asp85. Channel closure is
strongly coupled to deprotonation of Asp85 but less to the reprotonation of the Schiff base
which partially precedes the cytosolic conformational change. Azide accelerates channel
closure relative to the internal protontransfer steps. In addition to site-specific labelling, the
model-free analysis of cross-correlated spectral data can be extended to proteins containing
natural flurophores. We show for the cytoskeletal protein actin that the loss of liganddependent
static quenching of tryptophan emission during thermal unfolding correlates with
the loss of specific secondary structure monitored by FTIR spectroscopy. This approach
provides structural information on flavonoid binding to actin which has recently been shown
to affect actin conformational changes [2]. Further topological information can be obtained
from the emission wavelength of the tryptophans that become unquenched during ligand
dissociation. Fluorescence-IR-cross-correlation spectroscopy thus extends the IR-based
conformational analysis by site-specific information on local physical parameters (polarity,
electrostatics, etc.) which affect the emission of intrinsic or extrinsic fluorophores.
References
[1] N. Lehmann, U. Alexiev, K. Fahmy, J. Mol. Biol. 336, 1129–1141 (2007).
[2] M. Boehl, S. Tietze, A. Sokoll, S. Madathil, F. Pfennig, J. Apostolakis, K. Fahmy, H.-O. Gutzeit,
Biophys. J. 93, 2767-2780 (2007).
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