Wavelet Shrinkage to Resolve Single Molecule FRET Structural Landscape of the Isolated Ligand Binding Domain of the AMPA Receptor

Abstract

Single molecule fluorescence resonance energy transfer (smFRET) spectroscopy has provided significant advances in our understanding of the relationship between structure and function in biological systems. Currently, simplifications must be made for experimental systems, data analysis, and theoretical modeling because biomolecules often exhibit mechanistic or conformational heterogeneity. For example, it is often necessary to treat biomolecular processes as transitions between two well-define states (e.g. folded vs. unfolded) despite clear experimental evidence or theoretical predictions to the contrary. The conformations explored by the agonist binding domain of the α-amino-3-hydroxy-5-methyl-4-isoxazole propionate receptor represent such a system. The distribution of conformations in the raw data was so wide it was not possible to extract conformational details. By employing a model-free data analysis technique called wavelet shrinkage, it was determined that each protein form comprised multi-state, sequential equilibria. The results illustrate that the extent of activation is dependent not on a rigid closed cleft, but instead on the probability that a given subunit will occupy a closed cleft conformation, which in turn is determined by the range of states that the protein explores. Also, the results emphasize both the need for and the utility of advanced data processing techniques to quantify structure and dynamics in heterogeneous systems.