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18.02.14 10:36

Preis | Katherina Hemmen

Katherina Hemmen gewinnt auf dem Biophysical Society Meeting in San Francisco, USA, den Posterpreis der Fluorescence Subgroup.

Titel: Deciphering Folding Pathways of Phage T4 Lysozyme: Influence of Multiple Conformations

Abstract:

Understanding how a linear strand of amino acids folds into an active protein in the cell is still one of the challenges in biochemistry. Moreover, as quite a distinct amount of misfolded proteins are responsible for diseases, e. g. Alzheimer's. Thus, understanding the folding pathway, hence describing the energy landscape, of a protein is of critical importance. Here, we used the model enzyme lysozyme from the phage T4 (T4L), a simple two domain protein, which already exists in an equilibrium of at least three conformations in the folded state, a situation expected from most other proteins as well.

Thus, we created a set of double mutants of the cysteine-free pseudo-wild type by inserting an unnatural amino acid and a cysteine mutation, which builds up a network of distances spanning the enzyme. Later, we site-specifically labeled them using orthogonal chemistry with a Förster resonance energy transfer (FRET) dye pair. The unfolding process was monitored on the one hand using traditional ensemble methods like CD- spectroscopy and on the other hand with our fluorescence spectroscopic methods(ensemble steady-state fluorescence, ensemble time correlated single photon counting, single molecule fluorescence spectroscopy, filtered fluorescence correlation spectroscopy). Single molecule high precision FRET, with its high temporal and spatial resolution covering seven time decades, enables us to determine transition rates between conformational states in equilibrium and in denatured conditions. The combination of our fluorescence spectroscopic tool provides enough information to describe the energy landscape of the unfolding process of T4L and its paths to the unfolded conformations.

Verantwortlich für den Inhalt: E-Mail sendenProf. Dr. Claus Seidel