Submillisecond Dynamics of the NMDA Receptor

Abstract

Observation of single molecules has impacted the way we look at biomolecular machines. Particularly, because it gave us a tool to understand how biomachines move as they carry out specific functions. Single molecule fluorescence experiments, done in surface immobilized conditions or freely diffusing, take advantage of the fact that fluorescence occurs in the nanosecond timescale to map a wide range of biologically relevant dynamics, covering over 10 orders of magnitude in time without gaps. Single molecule Forster Resonance Energy Transfer (smFRET) experiments have evolved to the point where it is possible to work with complex systems including membrane proteins. However, many challenges have limited the impact on the field, mostly because there is the common conception that the obtained structural information is very limited. In addition, multiple experimental artifacts can complicate the interpretation of data. However, recent advances in smFRET experiments, particularly using Multiparameter Fluorescence Detection (MFD), have improved its accuracy and precision, to the point that it is possible to overcome this mindset. As an example, we use MFD to identify low populated conformational states of the ligand-binding domain of the N-methyl-D-aspartate (NMDA) receptors, quantify molecular dynamics in the sub millisecond regime and identify how conformational dynamics sheds light into the mechanism of partial agonism in the GluN1 subunit of the NMDA receptor.