My research focus is on developing and applying biophysical spectroscopy to study membrane protein regulations, with an emphasis on interpreting biochemical regulations (function) in terms of biophysical interactions (structural dynamics). Specifically, I am using time-resolved fluorescence resonance energy transfer (TRFRET), time-resolved phosphorescence anisotropy (TPA) and fluorescence microscopy to study SERCA and its regulator PLB, two transmembrane proteins that are responsible for 70% of the Ca uptake during cardiac muscle relaxation in the human heart.
Time-resolved FRET to resolve multiple structural states:
Our lab has shown that Ca and phosphorylation relieves SERCA inhibition without dissociating PLB, but rather inducing changes of structural dynamics within SERCA or PLB. In order to further elucidate the functional regulations in terms of physical interactions, I controlled the physical interaction between SERCA and PLB using charged lipids. Then used time-resolved FRET to resolve the structural states of the SERCA-PLB complex. PLB binds to SERCA at two structural states, corresponding to two dynamic states measured using EPR. The ordered membrane associated state is more inhibitory, whereas the disordered membrane disassociated state is less inhibitory.
TPA to resolve protein aggregation:
Previous TPA work from our lab also show that PLB induces SERCA aggregation and thus reduces its ATPase activity. However, previous work were based on ErITC, a phosphorescence derivative that binds to SERCA in the ATP binding pocket, thus significantly interferes with SERCA activity. I am labeling SERCA using ErIA in the native cardiac SR vesicles, and study the effects of PLB phosphorylation on SERCA aggregation. ErIA labeled SERCA is fully functional. TPA results show that phosphorylation of PLB increases rotational dynamics of SERCA, suggesting that PLB regulates SERCA aggregation in cardiac muscles. Unphosphorylated PLB induces SERCA aggregation and thus reduced its ATPase activity. Phosphorylation of PLB decreases SERCA aggregation and thus relieves the SERCA inhibition.
Polarized TIRF to resolve domain orientation:
I developed a polarized total internal reflection fluorescence microscopy to study the orientation of PLB labeled with bi-functional fluorescence label in supported lipid bilayer. SERCA binding lifts PLB cytoplasmic domain away from the membrane surface.
I am also managing the Biophysical Spectroscopy Facility (BSF). Please go to the BSF website and check our state of the art spectroscopic instruments. We are looking forward to sharing with you our knowledge in spectroscopy, and helping you to explore the fascinating protein world.