Kinetic studies of RRM dynamics using U1A protein as structural model

The RNA is an essential biopolymer that is typically bound to RNA binding proteins (RBPs). Because RNA-RBP complexes are vital, there has been considerable research to understand the molecular origins, but little is known about the kinetics of these interactions. The objective of this research project is to study the dynamics between RNA and the RNA recognition motif (RRM), which is the most common RBP domain. Our model system is the U1A protein and SL2 RNA. To study fast bimolecular kinetics, we are using time-resolved stopped-flow and laser temperature-jump (T-jump) methods.  From previous results, we developed a three step kinetic pathway. In this pathway, the first step of ~100 µs was assigned to an internal rearrangement of the complex from a tight to a loose complex, the second step of ~50 ms was assigned to the dissociation of the U1A-SL2 complex, and the third step of ~2 s was proposed to be a reorganization of the C-terminal helix of U1A protein after dissociation. This three step pathway was developed using a F56W construct. In order to support our hypothetical pathway we need to fully characterize the last step. We included a histidine quencher in two different positions of the C-terminal helix. This new constructs are D90HF56W and I94HF56W with ~60 fold reduction in binding affinity. Both positions interact in a different manner with the F56W and revealed different conformational changes in the quenching experiments that have to be study deeply in order to develop a complete kinetic pathway.