Theoretical Investigation of Kainate Receptor GluK2 and Channelrhodopsin-2: Structure and Mechanism

This work focuses on the structures and mechanisms of functions of two different proteins: the kainate receptor (GluK2) and the channelrhodopsin-2 (ChR2). GluK2 is a ligand-gated ionotropic glutamate receptor. Its channel opening and closing behaviors are controlled by the bound photoswitchable ligand. ChR2 is a lightgated ion channel, the opening of which is triggered directly by light. They are able to activate neuron cells or transfer signals between synapses upon illumination, and thus, control and monitor neuron cell activities in living tissues. This technique is called optogenetics. It has promising applications in studying neuronal diseases. The work starts with the structural model building of the proteins. The structure-function relationships of the proteins were investigated with multi-scale techniques, combining classic mechanical (MM) and quantum mechanical (QM) methods. The photochromic ligand gluazo is an azobenzene derivative. It alters the opening extent of the ion channel of GluK2 by changing the molecular configuration upon illumination. The protein response to gluazo isomerization is not fully understood. Additional gluazo binding mode, different from that in the crystal structure, was searched through the simulated annealing simulations. The preference of available binding modes was evaluated by free energy calculations. In the crystal structure the protein binds with the trans-gluazo. In order to obtain the complex of GluK2 binding with cis-gluazo and avoid the computational expensive high level quantum mechanical method, a forced switching protocol was employed. This method fulfills the current requirement by providing models for studying protein response to different ligand conformations. Extensive molecular dynamics (MD) simulations reveal that trans-gluazo and cis-gluazo have different interactions with the protein. This leads to the different efficiencies of the two ligands: trans-gluazo is a partial agonist, while cis-gluazo has a relatively lower efficacy than trans-gluazo. Although ChR2 and its various variants are widely applied in neuroscience, the investigation of their mechanisms of functions remains to be a hot topic. This study is about ChR2-C128T, which is a ChR2 mutant with slow photocycle kinetics. The mutation enlongates the life time of the open state. Starting from the mutant homology model, classical force field and combined QM/MM MD simulations were performed. Several structural motifs were investigated and analyzed. The active site of ChR2-C128T was found to be much more flexible than that of the well investigated bacteriorhodopisn (BR). All these motifs are highly correlated, which increases the complexity for studying ChR2. The rationality of the current models and samplings are proven by excitation energy calculations. The impact of the surrounding environment on the chromophore isomerization of ChR2 was investigated using the CASPT2//CASSCF/MM method (CASPT2: complete active space second order perturbation theory, CASSCF: complete active space self consistent field). The isomerization energy pathways of three models with different active site structural motifs were calculated. The study shows that, only one of the models can propagate an isomerization through conical intersection in a radiationless decay. The received isomerization product was analyzed by computing the infared spectrum that was then compared with the experimental results. The study shows the complexity of these light reactive proteins, while many different high level computational techniques are necessary to obtain findings about the structures and mechanisms.