Simulations of Fluorescent Probes Attached to SERCA

Abstract

Molecular simulations were performed with the aim to establish a more rigorous foundation for analyzing spectroscopic data on SERCA labeled with fluorescent probes. Site-specific labeling of a protein with fluorescent probes can provide insight into local structural dynamics, based on fluorescence quenching or anisotropy measurements. Distances to another label can be measured using FRET. From these experiments we will gain insight into the structure and dynamics of important biochemical states of SERCA. Cys674 in the P-domain of SERCA was labeled with the fluorescent probe IAEDANS to measure interdomain distances. The starting point for the molecular dynamics (MD) simulations was a new crystal structure of AEDANS-labeled SERCA, determined to 3.4Å resolution, which was sufficient to show the AEDANS label in close proximity to residues Arg615 and Arg620. To carry out MD simulations, we developed CHARMM force-field parameters for AEDANS and determined the orientation of the transition dipole moment from quantum chemistry calculations on ground and excited states of AEDANS. The transition dipole autocorrelation functions and reorientation times were calculated from the simulated trajectories and were shown to agree with experimental measurements by fluorescence anisotropy. FRET was measured using AEDANS as the donor and TNP-ADP bound in the nucleotide pocket as the acceptor. The interprobe distance R and the orientation factor κ2, determined from the simulations were used to calculate expected fluorescence lifetime changes due to FRET. The results show that we have established a reliable framework for both fluorescence experiments and simulations in this system. This work was supported by NIH (GM27906, AR007612) and the Minnesota Supercomputing Institute.