Dynamics of Firefly Luciferase Inhibition by General Anesthetics: Gaussian and Anisotropic Network Analyses

Abstract

The new crystal structures of the product-bound firefly luciferase combined with the previously determined substrate-free structures allow for a detailed analysis of the dynamics basis for the luciferase enzymatic activities. Using the Gaussian network model and the anisotropic network model, we show here that the superposition of the three slowest anisotropic network model modes, consisting of the bending, rotating, and rocking motions of the C-domain, accounts for large rearrangement of domains from the substrate-free (open) to product-bound (closed) conformation and thus constitutes a critical component of the enzyme's functions. The analysis also offers a unique platform to reexamine the molecular mechanism of the anesthetic inhibition of the firefly luciferase. Through perturbing the protein backbone network by introducing additional nodes to represent anesthetics, we found that the presence of two representative anesthetics, halothane and n-decanol, in different regions of luciferase had distinctively different effects on the protein's global motion. Only at the interface of the C- and N-domains did the anesthetics cause the most profound reduction in the overall flexibility of the C-domain and the concomitant increase in the flexibility of the loop, where the substitution of a conserved lysine residue was found experimentally to lead to >2-3 orders of magnitude reduction in activity. These anesthetic-induced dynamics changes can alter the normal function of the protein, appearing as an epiphenomenon of an "inhibition". The implication of the study is that a leading element for general anesthetic action on proteins is to disrupt the modes of motion essential to protein functions.

The role of proline substitutions within flexible regions on thermostability of luciferase

“…The high-resolution structure 1BA3 was chosen to be applied with MD simulation for two reasons. Firstly, the root mean-square deviation (RMSD) between 1LCI and 1BA3 is very small (b1 Å), consistent with the fact that 1BA3 was obtained by simply soaking the apoluciferase crystal transiently in a saturated bromoform solution [25]. Secondly, there are only two residues unresolved in 1BA3 while there are more than twenty residues missed in 1LCI.…”

The role of structured water in mediating general anesthetic action on α4β2 nAChR

“…First, because of its hydrophobic nature, site 4 closed had a negligible number of water molecules within a 7-A radius of the binding site before and after halothane binding. It is not surprising to see a relatively low probability (1/12, 8%) for this scenario, considering that anesthetics prefer an amphiphilic environment 20, 37–39. Second, the number of water molecules within 7 A from the center of site 6 closed and site 2 open (2/12, 17%) increased slightly.…”

“…Our recent MD simulations demonstrated that halothane altered the global motion of the α4β2 nAChR more in the open channel conformation than in the closed channel conformation,12, 13 suggesting that anesthetic inhibition to the α4β2 nAChR function might result from the anesthetic perturbation to the motion of the open channel. It is possible that water facilitates the anesthetic disturbance to the protein global motions that are essential for protein function 12, 18–20…”

Streptococcus mutans Inhibits Candida albicans Hyphal Formation by the Fatty Acid Signaling Molecule trans‐2‐Decenoic Acid (SDSF)

“…[34] Another type of inhibition was discovered unexpectedly some 40 years ago: general anesthetics, for example, N-decanol, are strong inhibitors of firefly luciferase. [35] Structurally related aliphatic compounds (dodecanol, dodecanoic acid, dodecylamine) are also able to completely quench the luminescence reaction. [36] We suggest that SDSF acts as a substrate analogue and inhibits the luciferase reaction.…”