Professor Don Lamb, Department of Chemistry, Ludwig-Maximilians-Universität, Munich, Germany

Measuring Protein Dynamics using Single Molecule FRET

Fluorescence spectroscopy and microscopy offers very sensitive, non-contact approaches to investigate biological systems. I will start by introducing 2-color and 3-color single-molecule Förster Resonance Energy Transfer (FRET) experiments and explain the insights we have obtained on the folding of a two-domain protein, the Maltose Binding Protein.
The two domains are discontinuous. By using 3c FRET, we could follow the order of domain folding and could show that the C-terminal domain can only fold after the N-terminal domain.
I will also present the DEEP-Learning Assisted Single molecule Image analysis (DEEP-LASI) approach we developed for performing an automated trace characterization and kinetic analysis of single-molecule FRET traces. This approach allows the analysis of single-molecule total internal reflection fluorescence microscopy experiments in minutes rather than days.