Paper Citing NAMD - Abstract
Najor, Matthew S.; Olsen, Kenneth W.; Graham, Daniel J.; de Freitas, Duarte Mota
Contribution of each Trp residue toward the intrinsic fluorescence of the G(i alpha 1) protein
PROTEIN SCIENCE, 23:1392-1402, OCT 2014
G(i alpha 1) is the inhibitory G-protein that, upon activation, reduces the activity of adenylyl cyclase. Comparison of the crystal structures of G(i alpha 1) bound to GDP center dot AMF or GTP gamma S with that of the inactive, GPD-bound protein indicates that a conformational change occurs in the activation step centered on three switch regions. The contribution of each tryptophan residue (W211 in the switch II region, W131 in the alpha-helical domain, and W258 in the GTPase domain) toward the intrinsic protein fluorescence was evaluated by using W211F, W131F, and W258F mutants. All three tryptophan residues contributed significantly toward the emission spectra regardless of the conformation. When activated by either GDP center dot AMF or GTP gamma S, the observed maximal-fluorescence scaled according to the solvent accessibilities of the tryptophan residues, calculated from molecular dynamics simulations. In the GDP center dot AMF and GTP gamma S, but not in the GDP, conformations, the residues W211 and R208 are in close proximity and form a pi-cation interaction that results in a red shift in the emission spectra of WT, and W131F and W258F mutants, but a blue shift for the W211F mutant. The observed shifts did not show a relationship with the span of the W211-R208 bridge, but rather with changes in the total interaction energies. Trypsin digestion of the active conformations only occurred for the W211F mutant indicating that the electrostatic pi-cation interaction blocks access to R208, which was consistent with the molecular dynamics simulations. We conclude that solvent accessibility and interaction energies account for the fluorescence features of G(i alpha 1).
