Dataset Information

Interpretation of spectroscopic data using molecular simulations for the secondary active transporter BetP.

ABSTRACT: Mechanistic understanding of dynamic membrane proteins such as transporters, receptors, and channels requires accurate depictions of conformational ensembles, and the manner in which they interchange as a function of environmental factors including substrates, lipids, and inhibitors. Spectroscopic techniques such as electron spin resonance (ESR) pulsed electron-electron double resonance (PELDOR), also known as double electron-electron resonance (DEER), provide a complement to atomistic structures obtained from x-ray crystallography or cryo-EM, since spectroscopic data reflect an ensemble and can be measured in more native solvents, unperturbed by a crystal lattice. However, attempts to interpret DEER data are frequently stymied by discrepancies with the structural data, which may arise due to differences in conditions, the dynamics of the protein, or the flexibility of the attached paramagnetic spin labels. Recently, molecular simulation techniques such as EBMetaD have been developed that create a conformational ensemble matching an experimental distance distribution while applying the minimal possible bias. Moreover, it has been proposed that the work required during an EBMetaD simulation to match an experimentally determined distribution could be used as a metric with which to assign conformational states to a given measurement. Here, we demonstrate the application of this concept for a sodium-coupled transport protein, BetP. Because the probe, protein, and lipid bilayer are all represented in atomic detail, the different contributions to the work, such as the extent of protein backbone movements, can be separated. This work therefore illustrates how ranking simulations based on EBMetaD can help to bridge the gap between structural and biophysical data and thereby enhance our understanding of membrane protein conformational mechanisms.

SUBMITTER: Leone V

PROVIDER: S-EPMC6400524 | biostudies-literature | 2019 Mar

REPOSITORIES: biostudies-literature

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Interpretation of spectroscopic data using molecular simulations for the secondary active transporter BetP.

Leone Vanessa V Waclawska Izabela I Kossmann Katharina K Koshy Caroline C Sharma Monika M Prisner Thomas F TF Ziegler Christine C Endeward Burkhard B Forrest Lucy R LR

The Journal of general physiology 20190206 3

Mechanistic understanding of dynamic membrane proteins such as transporters, receptors, and channels requires accurate depictions of conformational ensembles, and the manner in which they interchange as a function of environmental factors including substrates, lipids, and inhibitors. Spectroscopic techniques such as electron spin resonance (ESR) pulsed electron-electron double resonance (PELDOR), also known as double electron-electron resonance (DEER), provide a complement to atomistic structure ...[more]

PMID: 30728216

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Project description:Transport of ions and small molecules across the cell membrane against electrochemical gradients is catalyzed by integral membrane proteins that use a source of free energy to drive the energetically uphill flux of the transported substrate. Secondary active transporters couple the spontaneous influx of a "driving" ion such as Na+ or H+ to the flux of the substrate. The thermodynamics of such cyclical non-equilibrium systems are well understood, and recent work has focused on the molecular mechanism of secondary active transport. The fact that these transporters change their conformation between an inward-facing and outward-facing conformation in a cyclical fashion, called the alternating access model, is broadly recognized as the molecular framework in which to describe transporter function. However, only with the advent of high resolution crystal structures and detailed computer simulations, it has become possible to recognize common molecular-level principles between disparate transporter families. Inverted repeat symmetry in secondary active transporters has shed light onto how protein structures can encode a bi-stable two-state system. Based on structural data, three broad classes of alternating access transitions have been described as rocker-switch, rocking-bundle, and elevator mechanisms. More detailed analysis indicates that transporters can be understood as gated pores with at least two coupled gates. These gates are not just a convenient cartoon element to illustrate a putative mechanism but map to distinct parts of the transporter protein. Enumerating all distinct gate states naturally includes occluded states in the alternating access picture and also suggests what kind of protein conformations might be observable. By connecting the possible conformational states and ion/substrate bound states in a kinetic model, a unified picture emerges in which the symporter, antiporter, and uniporter functions are extremes in a continuum of functionality. As usual with biological systems, few principles and rules are absolute and exceptions are discussed as well as how biological complexity may be integrated in quantitative kinetic models that may provide a bridge from the structure to function.

Dataset Information

Interpretation of spectroscopic data using molecular simulations for the secondary active transporter BetP.

Publications

Interpretation of spectroscopic data using molecular simulations for the secondary active transporter BetP.

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