Long-Range Energy Transfer in Protein Megamolecules.

Area of Science:

• Biophysics
• Protein Engineering
• Molecular Biology

Background:

• Studying energy transfer in large protein complexes is synthetically challenging.
• Protein-based megamolecules offer a promising platform for controlled energy transfer studies.
• Previous limitations hindered investigations in the long length regime (5-20 nm).

Purpose of the Study:

• To investigate energy transfer in novel, precisely defined protein-based megamolecules.
• To explore the effect of tunable distances between donor and acceptor fluorescent proteins.
• To understand the relationship between megamolecule size and energy transfer efficiency.

Main Methods:

• Construction of monodisperse protein megamolecules (oligomers) with defined lengths and molecular weights.
• Incorporation of fluorescent proteins as donors and acceptors.
• Variation of inter-protein distances using cross-linker length and spacer proteins.
• Characterization using two-photon absorption, steady-state fluorescence, and fluorescence up-conversion spectroscopy.
• Rationalization of experimental results using molecular dynamics simulations.

Main Results:

• Strong coupling between donor and acceptor dipoles was observed via two-photon absorption.
• Steady-state fluorescence showed no effect of cross-linker length on energy transfer efficiency.
• Time-resolved fluorescence up-conversion revealed decreased energy transfer rates with increased cross-linker length.
• Molecular dynamics simulations explained discrepancies between steady-state and time-resolved measurements due to structural fluctuations.
• Energy transfer length increased with increasing megamolecule size.

Conclusions:

• Evidence for long-range energy transfer in large protein megamolecules was established.
• The study highlights the importance of considering structural dynamics in energy transfer measurements.
• Protein megamolecules provide a versatile system for fundamental studies of energy transfer processes.