dr. Ziad Ganim: Combined Single Molecule Force and Fluorescence Spectroscopy of the Unfolding and Refolding of the Green Fluorescent Protein

Abstract

We have performed combined single molecule force and fluorescence experiments on EGFP to understand folding/refolding pathways and how the secondary structure of the protein modulates optical properties of its chromophore. A new instrument was designed capable of such measurements without sacrificing force resolution and with minimal photodamage. During force-induced unfolding, there is evidence for two or more intermediates, and fluorescence is abruptly lost. EGFP refolding proceeds through a marginally stable and heterogeneous molten globule state. In the unfolded state, EGFP can switch into a refolding-incompetent state, which is evidence for a slow structural change such as proline isomerization. Fluorescence is only regained upon refolding to the most stable state. Photobleached EGFP molecules do not recover their fluorescence after unfolding and subsequent refolding, which provides evidence that photobleaching results from chemical damage rather than cis-trans isomerization or secondary structural change. These results provide a full understanding of the unfolding-refolding energy landscape of GFP, and how the conformational state affects the environment of the fluorophore. This picture reconciles three classes of previous experiments: : (1) bulk unfolding/refolding with fluorescence (2) single molecule force-unfolding and (3) single molecule fluorescence intensity fluctuations. These results are relevant to efforts developing EGFP as a genetically encoded force sensor, and its application in fluorescence recovery after photobleaching (FRAP) experiments and single molecule localization techniques.