Journal of Ameriacan Chemical Soceity (2018), DOI: 10.1021/jacs.8b08208


Taylor, E. (Taylor Elijah); Metcalf, K. (Metcalf, Kevin); Carlotti, B. (Carlotti, Benedetta); Lai, Cheng-Tsung; Modica, J. (Modica, Justin); Schatz, G. (Schatz, Gorge); Mrksich, M. (Mrksich, Milan); Goodson, T. (Goodson III, Theodore)


In this investigation, we report evidence for energy transfer in new protein-based megamolecules with tunable distances between donor and acceptor fluorescent proteins. The megamolecules used in this work are monodisperse oligomers, with molecular weights of ~100-300 kDa and lengths of ~5-20 nm, and are precisely defined structures of fusion protein building blocks and covalent crosslinkers. Such structures are promising because the study of energy transfer in protein complexes is usually difficult in this long length regime due to synthetic limitations. We incorporated fluorescent proteins into the megamolecule structure and varied the separation distance between donor and acceptor by changing the length of the crosslinker in dimer conjugates and inserting non-fluorescent spacer proteins to create oligomers. Two-photon absorption measurements demonstrated strong coupling between donor and acceptor dipoles in the megamolecules. For the dimer systems, no effect of the crosslinker length on energy transfer efficiency was observed with the steady-state fluorescence investigation. However, for the same dimer conjugates, energy transfer rates decreased upon increasing crosslinker length, as evaluated by fluorescence up-conversion. Molecular dynamics simulations were used to rationalize the results, providing quantitative agreement between measured and calculated energy transfer lengths for steady-state results, and showing that the differences between the time-resolved and steady-state measurements arise from the long timescale for large scale fluctuations in the megamolecule structure. Our data highlight the powerful sensitivity of time-resolved and non-linear spectroscopies to the megamolecule dynamics, even for megamolecules whose presumed sizes place them outside of the Förster regime.