Refining Protein Penetration into the Lipid Bilayer Using Fluorescence Quenching and Molecular Dynamics Simulations: The Case of Diphtheria Toxin Translocation Domain

Abstract

Dynamic disorder of the lipid bilayer presents a challenge for establishing structure–function relationships in membranous systems. The resulting structural heterogeneity is especially evident for peripheral and spontaneously inserting membrane proteins, which are not constrained by the well-defined transmembrane topology and exert their action in the context of intimate interaction with lipids. Here, we propose a concerted approach combining depth-dependent fluorescence quenching with Molecular Dynamics simulation to decipher dynamic interactions of membrane proteins with the lipid bilayers. We apply this approach to characterize membrane-mediated action of the diphtheria toxin translocation domain. First, we use a combination of the steady-state and time-resolved fluorescence spectroscopy to characterize bilayer penetration of the NBD probe selectively attached to different sites of the protein into membranes containing lipid-attached nitroxyl quenching groups. The constructed quenching profiles are analyzed with the Distribution Analysis methodology allowing for accurate determination of transverse distribution of the probe. The results obtained for 12 NBD-labeled single-Cys mutants are consistent with the so-called Open-Channel topology model. The experimentally determined quenching profiles for labeling sites corresponding to L350, N373, and P378 were used as initial constraints for positioning TH8–9 hairpin into the lipid bilayer for Molecular Dynamics simulation. Finally, we used alchemical free energy calculations to characterize protonation of E362 in soluble translocation domain and membrane-inserted conformation of its TH8–9 fragment. Our results indicate that membrane partitioning of the neutral E362 is more favorable energetically (by ~ 6 kcal/mol), but causes stronger perturbation of the bilayer, than the charged E362.

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Abbreviations

Tempo-PC:

1-Palmitoyl-2-oleoyl-sn-glycero-3-phospho(TEMPO)choline

n-Doxyl-PC:

1-Palmitoyl-2-stearoyl-(n-Doxyl)-sn-glycero-3-phosphocholine

POPC:

1-Palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine

POPG:

1-Palmitoyl-2-oleoyl-sn-glycero-3-phosphoglycerol

LUV:

Large unillamelar vesicles

MD:

Molecular dynamics

DA:

Distribution analysis

QP:

Quenching profile

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Acknowledgements

This research was supported in part by National Institutes of Health Grant P30-GM110761. A.K. also acknowledges support of Grant 0116U000835 of Ministry of Education and Science of Ukraine.

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Correspondence to Alexey S. Ladokhin.

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Kyrychenko, A., Lim, N.M., Vasquez-Montes, V. et al. Refining Protein Penetration into the Lipid Bilayer Using Fluorescence Quenching and Molecular Dynamics Simulations: The Case of Diphtheria Toxin Translocation Domain. J Membrane Biol 251, 379–391 (2018). https://doi.org/10.1007/s00232-018-0030-2

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Keywords

  • Diphtheria toxin
  • Depth-dependent fluorescence quenching
  • Distribution analysis
  • Alchemical free energy
  • Protonation