Unassisted Transport of N-Acetyl-l-tryptophanamide through Membrane: Experiment and Simulation of Kinetics

Abstract

Cellular transport machinery, such as channels and pumps, is working against the background of unassisted material transport through membranes. The permeation of a blocked tryptophan through a 1,2-Dioleoyl-sn-glycero-3-phosphocholine (DOPC) membrane is investigated to probe unassisted or physical transport. The transport rate is measured experimentally and modeled computationally. The time scale measured by Parallel Artificial Membrane Permeation Assay (PAMPA) experiments is ~8 h. Simulations with the Milestoning algorithm suggest Mean First Passage Time (MFPT) of ~4 h and the presence of a large barrier at the center of the bilayer. A similar calculation with the solubility-diffusion model yields MFPT of ~15 min. This permeation rate is nine orders of magnitude slower than the permeation rate of only a tryptophan side chain (computed by us and others). This difference suggests critical dependence of transport time on permeant size and hydrophilicity. Analysis of the simulation results suggests that the permeant partially preserves hydrogen bonding of the peptide backbone to water and lipid molecules even when it is moving closer to the bilayer center. As a consequence, defects of the membrane structure are developed to assist permeation.

Permeation of the three aromatic dipeptides through lipid bilayers: Experimental and computational study

“…Both phenylalanine and tyrosine have lower free energies inside the lipid than in water, while tryptophan has to overcome a significant free energy barrier in the membrane center. In studies of blocked tryptophan (Nacetyltryptophanamide or NATA), 43 a similar behavior was found as for the tryptophan sidechain, with NATA exhibiting a significantly higher barrier in the central region.…”

“…25 Previous studies of tryptophan using the more advanced method of milestoning report permeation times on the time scale of hours, which are also in agreement with experimental results. 43,44,85,86 Most other studies have also reported much larger and faster permeability coefficients using the inhomogeneous solubilitydiffusion model, which has been discussed in great detail in other works. 18,25,43,44,[87][88][89] Several assumptions underpin the solubility-diffusion model, including memoryless, diffusivetype motion along the reaction coordinate and the presence of only one slow variable describing the motion of the permeant-namely, the translocation along the membrane normal.…”

“…The inhomogeneous solubility diffusion model was then used to calculate the permeability coefficient P and mean first passage time (MFPT) ⟨τ⟩, 15,19,43,78 …”

“…14 Recent molecular dynamics studies have focused on a wide variety of molecules passively diffusing through membranes, such as water, [15][16][17] small molecules, [18][19][20][21][22][23] model drug compounds, 6,22,24,25 analgesics, [26][27][28] drug delivery systems, 29,30 dyes, 31,32 other lipids, 33,34 nanoparticles, [35][36][37] toxins, 38 small peptides, 39,40 and even transmembrane proteins. 41,42 However, only a handful of MD studies have examined amino acid-related systems and are confined to tryptophan, [43][44][45] arginine, 46,47 lysine, 47 and amino acid analogues. 48 In terms of potential of mean force (PMF), findings have consistently shown that small nonpolar molecules tend to be preferentially bound in the membrane center, while polar molecules tend to interact favorably with the lipid headgroups and experience a free energy barrier in the center.…”

Catch bond-like kinetics of helix cracking: Network analysis by molecular dynamics and Milestoning

“…42 Hence Milestoning makes it possible to obtain accurate kinetics starting from picoseconds and going forward to exceptionally long time scales (e.g., hours. 53 ). An advantage of the Markov State Model is that the book keeping is simpler and the number of states is smaller compared to the Milestoning method.…”