Impact of Urea on Detergent Micelle Properties

Co-solvents, such as urea, can entail drastic changes in the micellization behavior of detergents. We present a systematic quantification of the impact of urea on the critical micellar concentration, the micellization thermodynamics, and the micelle size in three homologous series of commonly used non-ionic alkyl detergents. To this end, we performed demicellization experiments by isothermal titration calorimetry and hydrodynamic size measurements by dynamic light scattering on alkyl maltopyranosides, cyclohexyl alkyl maltopyranosides, and alkyl glucopyranosides at urea concentrations of 0–8 M. For all detergents studied, we found that the critical micellar concentration increases exponentially because the absolute Gibbs free energy of micellization decreases linearly over the entire urea concentration range, as does the micelle size. In contrast, the enthalpic and entropic contributions to micellization reveal more complex, nonlinear dependences on urea concentration. Both free energy and size changes are more pronounced for long-chain detergents, which bury more apolar surface area upon micelle formation. The Gibbs free energy increments per methylene group within each detergent series depend on urea concentration in a linear fashion, although they result from the entropic term for alkyl maltosides but are of enthalpic origin for cyclohexyl alkyl maltosides. We compare our results to transfer free energies of amino acid side chains, relate them to protein-folding data, and discuss how urea-induced changes in detergent micelle properties affect <i>in vitro</i> investigations on membrane proteins

Extracavity Effect in Cyclodextrin/Surfactant Complexation

Cyclodextrin (CD) complexation is a convenient method to sequester surfactants in a controllable way, for example, during membrane-protein reconstitution. Interestingly, the equilibrium stability of CD/surfactant inclusion complexes increases with the length of the nonpolar surfactant chain even beyond the point where all hydrophobic contacts within the canonical CD cavity are saturated. To rationalize this observation, we have dissected the inclusion complexation equilibria of a structurally well-defined CD, that is, heptakis­(2,6-di-<i>O</i>-methyl)-β-CD (DIMEB), and a homologous series of surfactants, namely, <i>n</i>-alkyl-<i>N</i>,<i>N</i>-dimethyl-3-ammonio-1-propanesulfonates (SB3-<i>x</i>) with chain lengths ranging from <i>x</i> = 8 to 14. Thermodynamic parameters obtained by isothermal titration calorimetry and structural insights derived from nuclear magnetic resonance spectroscopy and molecular dynamics simulations revealed that, upon inclusion, long-chain surfactants with <i>x</i> = ≥10 extend beyond the canonical CD cavity. This enables the formation of hydrophobic contacts between long surfactant chains and the extracavity parts of DIMEB, which make additional favorable contributions to the stability of the inclusion complex. These results explain the finding that the stability of CD/surfactant inclusion complexes monotonously increases with the surfactant chain length even for long chains that completely fill the canonical CD cavity

Interactions of SDS with A8-35 monitored by isothermal titration calorimetry.

<p>Demicellization of SDS in the presence of various A8-35 concentrations: (A) Differential heating power, Δ<i>p</i>, versus time, monitored during the titration of 10 mM SDS and 20 µM A8-35 into 20 µM A8-35. (B) Normalized heats of reaction, <i>Q</i>_S, versus SDS concentration in the cell, [SDS], resulting from the dilution of micellar SDS solutions (10-13 mM) in the presence of A8-35 at A8-35 concentrations of 5 µM (△), 10 µM (O), 20 µM (◊), 40 µM (◃), 60 µM (▹), 80 µM (), 100 µM (☆), 120 µM (□), and 140 µM (+).</p

Sequence-Specific Dimerization of a Transmembrane Helix in Amphipol A8-35

<div><p>As traditional detergents might destabilize or even denature membrane proteins, amphiphilic polymers have moved into the focus of membrane-protein research in recent years. Thus far, Amphipols are the best studied amphiphilic copolymers, having a hydrophilic backbone with short hydrophobic chains. However, since stabilizing as well as destabilizing effects of the Amphipol belt on the structure of membrane proteins have been described, we systematically analyze the impact of the most commonly used Amphipol A8-35 on the structure and stability of a well-defined transmembrane protein model, the glycophorin A transmembrane helix dimer. Amphipols are not able to directly extract proteins from their native membranes, and detergents are typically replaced by Amphipols only after protein extraction from membranes. As Amphipols form mixed micelles with detergents, a better understanding of Amphipol-detergent interactions is required. Therefore, we analyze the interaction of A8-35 with the anionic detergent sodium dodecyl sulfate and describe the impact of the mixed-micelle-like system on the stability of a transmembrane helix dimer. As A8-35 may highly stabilize and thereby rigidify a transmembrane protein structure, modest destabilization by controlled addition of detergents and formation of mixed micellar systems might be helpful to preserve the function of a membrane protein in Amphipol environments.</p></div

Stoichiometry of GpA TM domain association.

<p>Energy transfer efficiency as a function of acceptor mole fraction in 5 µM (•) and 50 µM (○) APol (n = 2). Linear dependence of the energy transfer on the acceptor mole ratio demonstrates exclusive dimer formation of the GpA TM domain in APol.</p

Reversibility of GpA dimer dissociation by SDS addition to APol/GpA complexes.

<p>Dimer fractions shown without SDS and with 0.4 mM and 2 mM SDS added. The 2 mM SDS sample was then diluted to 0.4 mM SDS to demonstrate the reversibility of SDS-mediated dimer dissociation. The fraction dimer at 0.4 mM SDS after dilution from higher SDS concentrations is comparable to that observed at 0.4 mM SDS (n = 3).</p

Quantification of Millisecond Protein-Folding Dynamics in Membrane-Mimetic Environments by Single-Molecule Förster Resonance Energy Transfer Spectroscopy

An increasing number of membrane proteins in different membrane-mimetic systems have become accessible to reversible unfolding experiments monitored by well-established ensemble techniques. However, only little information is available about kinetic processes during membrane-protein folding, mainly because of experimental challenges and a lack of methods suitable for observing highly dynamic membrane proteins. Here, we present single-molecule Förster resonance energy transfer (smFRET) confocal spectroscopy as a powerful tool in kinetic studies of membrane-protein folding in membrane-mimetic environments. We have developed a rigorous workflow demonstrating how to identify and quantify such dynamic processes using a set of qualitative, semiquantitative, and quantitative analytical tools. Using this workflow, we analyzed urea-induced folding and unfolding experiments on the α-helical membrane protein Mistic in the presence of the zwitterionic detergent <i>n</i>-dodecylphosphocholine (DPC). We identified two-state interconversion dynamics on the millisecond time scale of a protein folding into and out of detergent micelles. Our results demonstrate that smFRET is a promising tool for probing the chemical physics of membrane-protein structure and dynamics in the complex and anisotropic environment of a hydrophilic/hydrophobic interface, providing insights into protein interconversion dynamics without the need and challenges of synchronization

Dissociation of the GpA TM domain in APol upon addition of SDS.

<p>Steady-state FRET measurements (n = 3) were performed at a fixed polymer (20 µM) to peptide (0.5 µM) ratio (40∶1). SDS was added to preformed APol/GpA complexes, and emission spectra were recorded at 25°C after incubation at 37°C. (A) Dimer fractions of the GpA TM domain determined by FRET efficiencies of the fluorescence emissions spectra plotted against SDS concentration (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0110970#pone.0110970.e005" target="_blank">eq. 3</a>). (B) Logarithm of the apparent GpA TM dissociation constants at increasing SDS concentration (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0110970#pone.0110970.e006" target="_blank">eq. 4</a>).</p

Association of the GpA TM domain in APol.

<p>FRET measurements (n = 3) were performed at increasing APol concentrations ranging from 5 to 75 µM, corresponding to APol/peptide ratios of 10∶1 to 150∶1. (A) Dimer fractions of the GpA TM domain determined by FRET efficiencies of the fluorescence emissions' spectra plotted against APol concentration (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0110970#pone.0110970.e005" target="_blank">eq. 3</a>). (B) Logarithm of the apparent GpA TM dissociation constants at increasing APol concentration (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0110970#pone.0110970.e006" target="_blank">eq. 4</a>).</p

Kinetics of the exchange of GpA TM peptides between APol aggregates.

<p>Donor- and acceptor-labelled GpA TM (each 0.25 µM) domains were separately solubilized in 20 µM APol at a final polymer/peptide ratio of 40∶1. (A) After incubation at 37°C, donor and acceptor were mixed, and emission spectra (normalized at 525 nm) were recorded every 10 min over a time period of 17 h at 25°C. (B) The energy transfer increased over hours to the level determined by steady-state FRET measurements due to mixing of donor- and acceptor-labelled peptides. (C) Energy transfer change due to peptide exchange in 5 mM DDM micelles was completed after some dozen minutes.</p