Fluorescence and Infrared Cross-Correlation Spectroscopy: A New Tool in Analysing Protein Conformational Coupling

The allosteric regulation of biomolecules such as enzymes or receptors is based on structural changes that are initiated at a ligand-binding site and become transmitted to a "distant" active site where enzymatic efficiency or interaction with effectors is altered. Understanding the molecular mechanisms of this long range coupling between distinct protein domains is crucial for many pharmacoligically relevant systems where the conformation of a target molecule has to be specifically affected by a designed ligand. We have developed a generalized multidimensional spectoscopic approach to investigate long range conformational coupling in proteins. It employs the integration of fluorescence emission and infrared absorption data recorded simultaneously from the same protein sample that undergoes conformational transitions in response to an external perturbation. Using attenuated total relfectance (ATR) Fourier-transform infrared (FTIR) difference spectroscopy, additional channels for excitation and detection of fluorescence where established by light guides positioned above the sample on the ATR crystal. Long range coupling in the signal transfer through rhodopsin has recently been identified by Fluorescence-IR-cross-correlation [1]. Using 2D-cross-correlation techniques, the kinetic asynchronicity of the emission from natural or artificial site-specific fluorophores relative to the secondary structure-sensitive IR-absorption bands can be determined. Thereby, IR absorptions can be identified in a model-free and unbiased way that can be assigned to secondary-strutural elements that become specifically stabilized by ligand interactions. Here, we demonstrate in a cytoskeletal protein the correlation of the loss of ligand-dependent static quenching of intrinsic tryptophan emission during thermal unfolding with the loss of structure monitored by FTIR spectroscopy. The high signal to noise ratio in 2D-correlation and the "synchronicity tagging" of the IR bands through their correlation with an independent monitor of ligand dissociation allows detecting ligand protein interactions with an accuracy that is not achieved by FTIR-spectroscopy alone. In addition, topological information can be obtained from the emission wavelength of the tryptophans that become gradually unquenched during temperature-induced ligand dissociation. Fluorescence-IR-cross-correlation spectroscopy thus extends the IR-based conformational analysis by the inclusion of site-specific information on local physical parameters (polarity, electrostatics, etc.) specifically affecting the emission of fluorophores. We show how this approach provides structural information on flavonoid binding to actin, a cytoskeletal and nuclear protein that has recently been shown to respond to the binding of these natural compounds by flavonoid-specific conformational changes [2].

Acknowledgement
We acknowledge financial support by the Deutsche Forschungsgemeinschaft to KF (grant 248/4)

References
[1] N. Lehmann, U. Alexiev, K. Fahmy, J. Mol. Biol. 336 (2007) 1129–1141.
[2] M. Boehl, S. Tietze, A. Sokoll, S. Madathil, F. Pfennig, J. Apostolakis, K. Fahmy, H.-O. Gutzeit,
Biophys. J. 93 (2007) 2767-2780.