Diffusion of glycerol through Escherichia coli aquaglyceroporin GlpF

Abstract

The glycerol uptake facilitator, GlpF, a major intrinsic protein found in Escherichia coli, selectively conducts water and glycerol across the inner membrane. The free energy landscape characterizing the assisted transport of glycerol by this homotetrameric aquaglyceroporin has been explored by means of equilibrium molecular dynamics over a timescale spanning 0.12 micros. To overcome the free energy barriers of the conduction pathway, an adaptive biasing force is applied to the glycerol molecule confined in each of the four channels. The results illuminate the critical role played by intramolecular relaxation on the diffusion properties of the permeant. These free energy calculations reveal that glycerol tumbles and isomerizes on a timescale comparable to that spanned by its adaptive-biasing-force-assisted conduction in GlpF. As a result, reorientation and conformational equilibrium of glycerol in GlpF constitute a bottleneck in the molecular simulations of the permeation event. A profile characterizing the position-dependent diffusion of the permeant has been determined, allowing reaction rate theory to be applied for investigating conduction kinetics based on the measured free energy landscape.

Figure 1

(Top) Free energy profile for glycerol conduction in the GlpF homotetramer obtained from equilibrium ABF simulations totaling 0.12 μs of sampling, via an integration of the force exerted on glycerol along the z-direction of Cartesian space. This potential of mean force corresponds to an average of the individual free energy profiles determined in the four channels forming GlpF. (Inset) Z-dependent diffusion of glycerol in GlpF was derived from additional simulations, wherein the permeant is confined in a harmonic potential. (Bottom) Cross-sectional view of a GlpF monomer. Pore-lining residues are rendered as a smooth surface, whereas a cartoon representation is used to depict the rest of the protein. The residues forming the SF and the water molecules contained in the pore are drawn in a licorice representation. The glycerol molecule located at the SF is highlighted in a space-filling representation. Image rendering was done with VMD .

Figure 2

Energetics deduced from incomplete sampling of glycerol transport across the SF region of GlpF. One trajectory captures a local minimum (a) mirrored in a typical conformation of the permeant (b). Another trajectory samples a monolithic barrier (c) corresponding to an inadequate orientation and conformation of glycerol (d). (b and d) Glycerol is shown in space-filling representation, and protein side chains (Trp⁴⁸, Phe²⁰⁰, Arg²⁰⁶) are shown in licorice representation. Carbon atoms are shown in light blue, nitrogen atoms are in dark blue, hydrogen atoms are in white, and oxygen atoms are in red, except for atom O1 of glycerol, which is shown in orange. Image rendering (b and d) was done with VMD (41).

Figure 3

(Top) Conformational and orientational preference of glycerol in the GlpF homotetramer, as a function of the reaction coordinate, z: Population of gauche-gauche (dark, solid line), gauche-anti (light, solid line), and anti-anti (dark, dashed line) conformations. (Bottom) Total molecular dipole moment of glycerol (dark, solid line) and its projection along the z-direction of Cartesian space (light, solid line), averaged over the four channels of GlpF.

Figure 4

Conformational equilibrium of glycerol in the SF region of the GlpF homotetramer: gauche-gauche (a), gauche-anti (b), and anti-anti (c) conformations. Image rendering was done with VMD .

Figure 5

Evolution of the number of hydrogen bonds as a function of the reaction coordinate, z. (Top) Hydrogen-bond acceptors; (bottom) hydrogen-bond donors. Asn⁶⁸ and Asn²⁰³ belong to the NPA motif. Arg²⁰⁶ is able to form two hydrogen bonds simultaneously, hence an average number greater than unity.

Figure 6

Conformational and orientational relaxation of glycerol in the GlpF homotetramer: Time-evolution of the two torsional angles of glycerol confined in the SF (a) and in bulk water (b). Time-evolution of the orientation of glycerol in the SF (c) and in the periplasmic vestibule (d). Monomer 1 (dark, solid line) versus monomer 2 (light, solid line).