Contribution of each Trp residue toward the intrinsic fluorescence of the G_iα1 protein

Abstract

G ia1 is the inhibitory G-protein that, upon activation, reduces the activity of adenylyl cyclase. Comparison of the crystal structures of G ia1 bound to GDP•AMF or GTPcS 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 a-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•AMF or GTPcS, the observed maximal-fluorescence scaled according to the solvent accessibilities of the tryptophan residues, calculated from molecular dynamics simulations. In the GDP•AMF and GTPcS, but not in the GDP, conformations, the residues W211 and R208 are in close proximity and form a p-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 p-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 ia1 .

Parametric models to compute tryptophan fluorescence wavelengths from classical protein simulations

“…Figures A and B show the computed emission spectra of Parvalbumin, Human Serum Albumin, and Glucagon, computed using Model 2 and Model 4, respectively. As expected, the variability of the emission wavelengths predicted by the simulations is smaller than that observed experimentally . However, the experimental broadening can be reasonably fit considering a broadening of 15 nm.…”

“…For instance, the analysis of the transition energy for the identification of the correct states to compute the energy of vertical transitions requires specific expertise. On the other side, previous studies reported that the emission wavelengths of Trp residues can be correlated in specific cases to simple structural parameters, as the solvent exposure of specific atoms, or the presence of local hydrogen bonds …”

Folding of Gα Subunits: Implications for Disease States

“…The W211 residue in WT G iα1 has been shown to have the largest difference in solvent accessibility between the inactive and active conformations and therefore contributes the most toward Δ F max . 19 Not surprisingly, for the W211F mutant of G iα1 , no Δ F max is observed ( Figure 7 B). Similarly, the W234 residue in G sα likely undergoes a similar large decrease in solvent accessibility during the course of the conformational change, as evidenced by the negligible Δ F max observed in the W234F mutant ( Figure 7 A).…”

“…The amino acid F was chosen as a replacement for W because of its similar structure and size characteristics as well as low quantum yield and distinct λ max values. 19 , 25 …”

“…The simulations were done using procedures previously described. 19 Unrestrained dynamics was run for 14 ns before the data were acquired for an additional 1 ns. The simulations were done at 37 °C (310 K) and 50 °C (328 K).…”