TOAC Spin Labels in the Backbone of Alamethicin: EPR Studies in Lipid Membranes

Max-Planck-Institut für Biophysikalische Chemie, Abteilung Spektroskopie, Göttingen, Germany.
Biophysical Journal (Impact Factor: 3.97). 01/2007; 92(2):473-81. DOI: 10.1529/biophysj.106.092775
Source: PubMed


Alamethicin is a 19-amino-acid residue hydrophobic peptide that produces voltage-dependent ion channels in membranes. Analogues of the Glu(OMe)(7,18,19) variant of alamethicin F50/5 that are rigidly spin-labeled in the peptide backbone have been synthesized by replacing residue 1, 8, or 16 with 2,2,6,6-tetramethyl-piperidine-1-oxyl-4-amino-4-carboxyl (TOAC), a helicogenic nitroxyl amino acid. Conventional electron paramagnetic resonance spectra are used to determine the insertion and orientation of the TOAC(n) alamethicins in fluid lipid bilayer membranes of dimyristoyl phosphatidylcholine. Isotropic (14)N-hyperfine couplings indicate that TOAC(8) and TOAC(16) are situated in the hydrophobic core of the membrane, whereas the TOAC(1) label resides closer to the membrane surface. Anisotropic hyperfine splittings show that alamethicin is highly ordered in the fluid membranes. Experiments with aligned membranes demonstrate that the principal diffusion axis lies close to the membrane normal, corresponding to a transmembrane orientation. Combination of data from the three spin-labeled positions yields both the dynamic order parameter of the peptide backbone and the intramolecular orientations of the TOAC groups. The latter are compared with x-ray diffraction results from alamethicin crystals. Saturation transfer electron paramagnetic resonance, which is sensitive to microsecond rotational motion, reveals that overall rotation of alamethicin is fast in fluid membranes, with effective correlation times <30 ns. Thus, alamethicin does not form large stable aggregates in fluid membranes, and ionic conductance must arise from transient or voltage-induced associations.

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Available from: Derek Marsh, Apr 25, 2014
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    • "Other studies detected both orientations depending on peptide concentration and hydration [20] [21] [22]. Conflicting findings have also been reported on the aggregation state of ALM in membranes in the absence of voltage, with some studies finding predominantly monomers [23] [24] [25], while others detected oligomers [26] [27] [28] [29] [30], with aggregation diminishing at higher temperatures [31]. Low-resolution information on the structure of the ALM pore has been obtained by neutron scattering. "
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    ABSTRACT: The structure and energetics of alamethicin Rf30 monomer to nonamer in cylindrical pores of 5 to 11Å radius are investigated using molecular dynamics simulations in an implicit membrane model that includes the free energy cost of acyl chain hydrophobic area exposure. Stable, low energy pores are obtained for certain combinations of radius and oligomeric number. The trimer and the tetramer formed 6Å pores that appear closed while the larger oligomers formed open pores at their optimal radius. The hexamer in an 8Å pore and the octamer in an 11Å pore give the lowest effective energy per monomer. However, all oligomers beyond the pentamer have comparable energies, consistent with the observation of multiple conductance levels. The results are consistent with the widely accepted "barrel-stave" model. The N terminal portion of the molecule exhibits smaller tilt with respect to the membrane normal than the C terminal portion, resulting in a pore shape that is a hybrid between a funnel and an hourglass. Transmembrane voltage has little effect on the structure of the oligomers but enhances or decreases their stability depending on its orientation. Antiparallel bundles are lower in energy than the commonly accepted parallel ones and could be present under certain experimental conditions. Dry aggregates (without an aqueous pore) have lower average effective energy than the corresponding aggregates in a pore, suggesting that alamethicin pores may be excited states that are stabilized in part by voltage and in part by the ion flow itself.
    Biochimica et Biophysica Acta 09/2013; 1838(5). DOI:10.1016/j.bbamem.2013.09.012 · 4.66 Impact Factor
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    • "A large variety of spin label building blocks for Boc-or Fmoc-based step-by-step peptide synthesis either on a solid support (SPPS) (Merrifield, 1963) or in solution have been synthesized (Barbosa et al., 1999; Elsässer et al., 2005). Being the most popular one, the paramagnetic α-amino acid TOAC (4-amino-1-oxyl-2,2,6,6,-tetramethyl-piperidine-4- carboxylic acid) (Rassat and Rey, 1967) is characterized by only one degree of freedom, the conformation of the six-membered ring (Fig. 2) The nitroxide is rigidly coupled to the peptide backbone, thereby providing the possibility to obtain direct information about the orientation of secondary structure elements, and has for example been used to study the secondary structure of small peptides in liquid solution (Anderson et al., 1999; Hanson et al., 1996; Marsh et al., 2007), and has also been successfully incorporated into the α-melanocyte stimulating hormone without loss of function (Barbosa et al., 1999). "
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    ABSTRACT: The function of a living cell, independent of we are talking about a prokaryotic single-cellular organism or a cell in the context of an complex organism like a human, depends on intricate and balanced interaction between its components. Proteins are playing a central role in this complex cellular interaction network: Proteins interact with nucleic acids, with membranes of all cellular compartments, and, what will be in the focus of this article, with other proteins. Proteins interact to form functional units, to transmit signals for example perceived at the surface of the cell to cytoplasmic or nuclear components, or to target them to specific locations. Thus, the study of protein-protein interactions on the molecular level provides insights into the basic functional concepts of living cells and emerged as a wide field of intense research, steadily developing with the introduction of new and refined biochemical and biophysical methods. Nowadays there is a vast of methods available to study the interaction between proteins. On the biochemical level mutational studies, crosslinking experiments and chromatographic techniques provide means to identify and characterize the interfaces on the protein surface where interaction takes place. Biophysical methods include calorimetric techniques, fluorescence spectroscopy and microscopy, and "structural techniques" like X-ray crystallography, (cryo-) electron microscopy, NMR spectroscopy, FRET spectroscopy, and EPR spectroscopy on spin labelled proteins.Site-directed spin labeling (SDSL) (Altenbach et al., 1989a, 1990) in combination with electron paramagnetic resonance (EPR) spectroscopy has emerged as a powerful tool to investigate the structural and the dynamical aspects of biomolecules, under conditions close to physiological i.e. functional state of the system under exploration. The technique is applicable to soluble molecules and membrane bound proteins either solubilised in detergent or embedded in a lipid bilayer. Therein, the size and the complexity of the system under investigation is almost arbitrary (reviewed in Bordignon & Steinhoff, 2007; Hubbell et al., 1996; Hubbell et al., 1998; Klare & Steinhoff, 2009; Klug & Feix, 2007). Especially with respect to protein-protein interactions SDSL EPR can provide a vast amount of information about almost all aspects of this interaction. Spin labeling approaches can provide detailed information about the binding interface not only on the structural level but also give insights into kinetic and thermodynamic aspects of the interaction. EPR also allows determination of distances between pairs of spin labels in the range from ~ 10-80 Å with accuracies down to less than 1 Å, thereby covering a range of sized including also large multi-domain proteins and protein complexes. This chapter will give an introduction into the technique of SDSL EPR spectroscopy exemplified with data from studies on the photoreceptor/transducer-complex NpSRII/NpHtrII, followed by a number of recent examples from the literature where protein-protein interactions have been studied using this technique.
    03/2012; Intech., ISBN: 978-953-51-0244-1
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    • "itored by means of ED - EPR at 77 K , indicated a greater tendency to form specific oligomers upon interaction of the peptides with DOPC than with DMPC bilayers ( Bartucci et al . 2009 ) . Based on CW - EPR spectra of aligned samples and from the relative polarity of the environments experienced by the different TOAC positions ( 1 , 8 , and 16 ) , Marsh et al . ( 2007b ) concluded that [ Glu ( OMe ) 7 , 18 , 19 ] alamethicin adopts a trans - membrane orientation in DMPC fluid bilayers . The authors used the combined order parameters for the different positions to determine the tilt angle of the peptide long axis relative to the bilayer normal . Effective tilt angles of 17 – 27 º were found over the te"
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    ABSTRACT: We review work on the paramagnetic amino acid 2,2,6,6-tetramethyl-N-oxyl-4-amino-4-carboxylic acid, TOAC, and its applications in studies of peptides and peptide synthesis. TOAC was the first spin label probe incorporated in peptides by means of a peptide bond. In view of the rigid character of this cyclic molecule and its attachment to the peptide backbone via a peptide bond, TOAC incorporation has been very useful to analyze backbone dynamics and peptide secondary structure. Many of these studies were performed making use of EPR spectroscopy, but other physical techniques, such as X-ray crystallography, CD, fluorescence, NMR, and FT-IR, have been employed. The use of double-labeled synthetic peptides has allowed the investigation of their secondary structure. A large number of studies have focused on the interaction of peptides, both synthetic and biologically active, with membranes. In the latter case, work has been reported on ligands and fragments of GPCR, host defense peptides, phospholamban, and β-amyloid. EPR studies of macroscopically aligned samples have provided information on the orientation of peptides in membranes. More recent studies have focused on peptide-protein and peptide-nucleic acid interactions. Moreover, TOAC has been shown to be a valuable probe for paramagnetic relaxation enhancement NMR studies of the interaction of labeled peptides with proteins. The growth of the number of TOAC-related publications suggests that this unnatural amino acid will find increasing applications in the future.
    Biophysical Reviews 03/2012; 4(1):45-66. DOI:10.1007/s12551-011-0064-5
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