Membrane protein dynamics in different environments: simulation study of the outer membrane protein X in a lipid bilayer and in a micelle

Choutko, Alexandra; Glättli, Alice; Fernández, César Fernández; Hilty, Christian; Wüthrich, Kurt; Gunsteren, Wilfred F. van

doi:10.1007/s00249-010-0626-7

Cited by 13 publications

(9 citation statements)

References 72 publications

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“…7A, the magnitude of the root mean square fluctuation (RMSF) values of the Cα atoms in both simulations resembles the pattern of the heteronuclear NOE values of OmpX in nanodiscs. This finding corroborates the NMR NOE, chemical shift and dynamics data and indicates, in line with previous results 26 , that loops 3 and 5 in OmpX do not adopt a well-ordered secondary structure in solution. The extracellular extruding β-strands observed in the crystal structure also exhibited large RMSF values in these simulations emphasizing the flexibility of this region.…”

Section: Resultssupporting

confidence: 93%

Optimized Phospholipid Bilayer Nanodiscs Facilitate High-Resolution Structure Determination of Membrane Proteins

et al. 2013

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Structural studies of membrane proteins are still hampered by difficulties of finding appropriate membrane mimicking media that maintain protein structure and function. Phospholipid nanodiscs seem promising to overcome the intrinsic problems of detergent containing environments. While nanodiscs can offer a near native environment, the large particle size complicates their routine use in the structural analysis of membrane proteins by solution NMR. Here, we introduce nanodiscs assembled from shorter ApoA-I protein variants that are of markedly smaller diameter and show that the resulting discs provide critical improvements for the structure determination of membrane proteins by NMR. Using the bacterial outer membrane protein OmpX as an example, we demonstrate that the combination of small nanodisc size, high deuteration levels of protein and lipids and the use of advanced non-uniform NMR sampling methods enable the NMR resonance assignment as well as the high-resolution structure determination of polytopic membrane proteins in a detergent-free, near-native lipid bilayer setting. By applying this method to bacteriorhodopsin we show that our smaller nanodiscs can also be beneficial for the structural characterization of the important class of seven-transmembrane helical proteins. Our set of engineered nanodiscs of subsequently smaller diameters can be used to screen for optimal NMR spectral quality for small to medium sized membrane proteins while still providing a functional environment. In addition to their key improvements for de novo structure determination, due to their smaller size these nanodiscs enable the investigation of interactions between membrane proteins and their (soluble) partner proteins, unbiased by the presence of detergents that might disrupt biological relevant interactions.

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Section: Resultssupporting

confidence: 93%

Optimized Phospholipid Bilayer Nanodiscs Facilitate High-Resolution Structure Determination of Membrane Proteins

et al. 2013

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“…In the case of recombinant-expressed peptides, weak alignment of peptides using acrylamide gels has been used to obtain residual dipolar couplings for orientational-dependent restraints (151–153). The positioning of peptides with respect to micelle surfaces has been inferred by paramagnetic relaxation enhancement mapping (34, 38, 147, 154, 155), water exposure measurements based on exchange peaks with the water signal (156–158), detection of NOEs between protonated micelles and peptides (159–162), or saturation transfer techniques (163–165). …”

Section: Nmr Spectroscopic Approaches To Study Ampsmentioning

confidence: 99%

On the Role of NMR Spectroscopy for Characterization of Antimicrobial Peptides

Porcelli

Ramamoorthy

Bárány

et al. 2013

Methods in Molecular Biology

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Summary Antimicrobial peptides (AMPs) provide a primordial source of immunity, conferring upon eukaryotic cells resistance against bacteria, protozoa, and viruses. Despite a few examples of anionic peptides, AMPs are usually relatively short positively charged polypeptides, consisting of a dozen to about a hundred amino acids, and exhibiting amphipathic character. Despite significant differences in their primary and secondary structures, all AMPs discovered to date share the ability to interact with cellular membranes, thereby affecting bilayer stability, disrupting membrane organization, and/or forming well-defined pores. AMPs selectively target infectious agents without being susceptible to any of the common pathways by which these acquire resistance, thereby making AMPs prime candidates to provide therapeutic alternatives to conventional drugs. However, the mechanisms of AMP actions are still a matter of intense debate. The structure-function paradigm suggests that a better understanding of how AMPs elicit their biological functions could result from atomic resolution studies of peptide-lipid interactions. In contrast, more strict thermodynamic views preclude any roles for three-dimensional structures. Indeed, the design of selective AMPs based soley on structural parameters has been challenging. In this chapter, we will focus on selected AMPs for which studies on the corresponding AMP-lipid interactions have helped reach an understanding of how AMP effects are mediated. We will emphasize the roles of both liquid- and solid-state NMR spectroscopy for elucidating the mechanisms of action of AMPs.

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“…7 and 9). This feature has not been evident in previous simulations of PagP (Cox and Sansom, 2009) or other membrane proteins (Rodríguez-Ropero and Fioroni, 2012;Friemann et al, 2009;Patargias et al, 2005;Bond and Sansom, 2003;Choutko et al, 2011;Sands et al, 2006;Psachoulia et al, 2006;Bond et al, 2004;Böckmann and Caflisch, 2005;, in whole or in part because of the small number of detergent and/or protein molecules in previous simulations. Unexpectedly, the above results suggest that these interactions can mediate protein aggregation at sufficiently high detergent concentration.…”

Section: Discussionmentioning

confidence: 75%

“…Since then, simulations have been used to characterize the conformations and interactions of many other proteins and peptides in various detergents. While most of these studies have been initiated with a preformed detergent micelle (Wymore and Wong, 1999;Rodríguez-Ropero and Fioroni, 2012;Friemann et al, 2009;Cuthbertson et al, 2006;Lagüe et al, 2005;Khandelia and Kaznessis, 2005a,b;Löw et al, 2008;Langham et al, 2007;Chevalier et al, 2006;Patargias et al, 2005;Bond and Sansom, 2003;Khao et al, 2011;Renthal et al, 2011;Cox and Sansom, 2009;Choutko et al, 2011;Krishnamani and Lanyi, 2012;Sands et al, 2006;Psachoulia et al, 2006), simulations have also evaluated the self-assembly of detergents around a protein or peptide using atomistic (Psachoulia et al, 2006;Bond et al, 2004;Böckmann and Caflisch, 2005;Braun et al, 2004;Jalili and Akhavan, 2011) and coarse-grained (Jalili and Akhavan, 2011;Friedman and Caflisch, 2011;Bond et al, 2007; models. These simulation systems, however, have almost exclusively contained a single protein molecule or preformed dimer (or crystal lattice ).…”

Section: Introductionmentioning

confidence: 99%

Detergent-mediated protein aggregation

Neale

Ghanei

Holyoake

et al. 2013

Chemistry and Physics of Lipids

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Because detergents are commonly used to solvate membrane proteins for structural evaluation, much attention has been devoted to assessing the conformational bias imparted by detergent micelles in comparison to the native environment of the lipid bilayer. Here, we conduct six 500-ns simulations of a system with >600,000 atoms to investigate the spontaneous self assembly of dodecylphosphocholine detergent around multiple molecules of the integral membrane protein PagP. This detergent formed equatorial micelles in which acyl chains surround the protein's hydrophobic belt, confirming existing models of the detergent solvation of membrane proteins. In addition, unexpectedly, the extracellular and periplasmic apical surfaces of PagP interacted with the headgroups of detergents in other micelles 85 and 60% of the time, respectively, forming complexes that were stable for hundreds of nanoseconds. In some cases, an apical surface of one molecule of PagP interacted with an equatorial micelle surrounding another molecule of PagP. In other cases, the apical surfaces of two molecules of PagP simultaneously bound a neat detergent micelle. In these ways, detergents mediated the non-specific aggregation of folded PagP. These simulation results are consistent with dynamic light scattering experiments, which show that, at detergent concentrations ≥600 mM, PagP induces the formation of large scattering species that are likely to contain many copies of the PagP protein. Together, these simulation and experimental results point to a potentially generic mechanism of detergent-mediated protein aggregation.

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Membrane protein dynamics in different environments: simulation study of the outer membrane protein X in a lipid bilayer and in a micelle

Cited by 13 publications

References 72 publications

Optimized Phospholipid Bilayer Nanodiscs Facilitate High-Resolution Structure Determination of Membrane Proteins

Optimized Phospholipid Bilayer Nanodiscs Facilitate High-Resolution Structure Determination of Membrane Proteins

On the Role of NMR Spectroscopy for Characterization of Antimicrobial Peptides

Detergent-mediated protein aggregation

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