Nonlinear spectroscopy of bound chromophores on peptides

Preface

Interdisciplinary cooperation with the Plant Physiology group at the Institute for Biochemistry and Biology:

Topics

• Investigation of electron and energy transfer processes in pigments, e.g. the light-harvesting complexes of the plant photosynthetic apparatus by use of transient spectroscopy
• Our plans: Exploring changes in the 3D-structure of proteins, i.e. conformational changes and folding processes and states with ESA techniques.
  Motivation: Changes in conformation are essential processes in nature, e.g. in enzymatic reactions. Misfolding of proteins can lead to aggregations as e.g. in the prion protein. The native fold is a prerequisite for a functional interaction with other proteins.

Results:

The model system of the protein myoglobin (red muscle dye, oxygen carrier, see ribbon model) and the fluorescent dye fluorescein isothiocyanate (FITC) was synthesized, purified and characterized by different methods.

Synthesis: The conjugation products were chemically heterogeneous with respect to their number and location of fluorophore per/on the protein (as shown by UV/VIS spectroscopy, HPLC, MALDI-TOF mass spectrometry). Preferred binding sites were the lysine residues 45, 62, 87, 98, 145 and the N-terminus (see backbone picture).

ESA spectroscopy: ESA spectra of native conjugates exhibit two maxima (around 405 and 435 nm). Both the unbound fluorophore and the denatured conjugate have a single maximum at 410 nm (see graph). Thus, in the native conjugate the population of bound fluorophore is also altered photophysically, and is also heterogeneous as shown by comparison with fluorescence decay data.

Conclusion:

Extrinsic fluorophores are suitable reporters of their protein environment. This is reflected in their ESA spectra.