Jan Raap

Structure and Alignment of the Membrane-Associated Peptaibols Ampullosporin A and Alamethicin by Oriented 15N and 31P Solid-State NMR Spectroscopy

Ampullosporin A and alamethicin are two members of the peptaibol family of antimicrobial peptides. These compounds are produced by fungi and are characterized by a high content of hydrophobic amino acids, and in particular the alpha-tetrasubstituted amino acid residue ?-aminoisobutyric acid. Here ampullosporin A and alamethicin were uniformly labeled with (15)N, purified and reconstituted into oriented phophatidylcholine lipid bilayers and investigated by proton-decoupled (15)N and (31)P solid-state NMR spectroscopy. Whereas alamethicin (20 amino acid residues) adopts transmembrane alignments in 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) or 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC) membranes the much shorter ampullosporin A (15 residues) exhibits comparable configurations only in thin membranes. In contrast the latter compound is oriented parallel to the membrane surface in 1,2-dimyristoleoyl-sn-glycero-3-phosphocholine and POPC bilayers indicating that hydrophobic mismatch has a decisive effect on the membrane topology of these peptides. Two-dimensional (15)N chemical shift -(1)H-(15)N dipolar coupling solid-state NMR correlation spectroscopy suggests that in their transmembrane configuration both peptides adopt mixed alpha-/3(10)-helical structures which can be explained by the restraints imposed by the membranes and the bulky alpha-aminoisobutyric acid residues. The (15)N solid-state NMR spectra also provide detailed information on the helical tilt angles. The results are discussed with regard to the antimicrobial activities of the peptides.

PULSED ELDOR IN SPIN-LABELED POLYPEPTIDES

Abstract The pulsed electron–electron double-resonance (PELDOR) technique was applied to obtain information about the structure of the synthetic polypeptide–biradical in a frozen glassy solution. From the concentration dependence of the PELDOR signal, the effects of intermolecular and intramolecular interactions were separated. It was found that the intramolecular dipole–dipole interactions in the biradical peptide led to the modulation effects in the PELDOR signal decay. This may be attributed to the existence of a conformational population having a distance between the two unpaired electrons of ∼20 A with a distribution of (∼2 A). Its fraction is estimated as about 25%.

Asymmetric binding of the primary acceptor quinone in reaction centers of the photosynthetic bacterium Rhodobacter sphaeroides R26, probed with Q-band (35 GHz) EPR spectroscopy

The reaction center (RC)‐bound primary acceptor quinone QA of the photosynthetic bacterium Rhodobacter sphaeroides R26 functions as a one‐electron gate. The radical anion Q•− A is proposed to have an asymmetric electron distribution, induced by the protein environment. We replace the native ubiquinone‐10 (UQ10) with specifically 13C‐labelled UQ10, and use Q‐band (35 GHz) EPR spectroscopy to investigate this phenomenon in closer detail. The direct observation of the 13C‐hyperfine splitting of the g z‐component of UQ10•− A in the RC and in frozen isopropanol shows that the electron spin distribution is symmetric in the isopropanol glass, and asymmetric in the RC. Our results allow qualitative assessment of the spin and charge distribution for Q•− A in the RC. The carbonyl oxygen of the semiquinone anion nearest to the S = 2 Fe2+‐ion and QB is shown to acquire the highest (negative) charge density.

Spatial structure of zervamicin IIB bound to DPC micelles: implications for voltage-gating.

Zervamicin IIB is a 16-amino acid peptaibol that forms voltage-dependent ion channels with multilevel conductance states in planar lipid bilayers and vesicular systems. The spatial structure of zervamicin IIB bound to dodecylphosphocholine micelles was studied by nuclear magnetic resonance spectroscopy. The set of 20 structures obtained has a bent helical conformation with a mean backbone root mean square deviation value of approximately 0.2 A and resembles the structure in isotropic solvents (Balashova et al., 2000. NMR structure of the channel-former zervamicin IIB in isotropic solvents. FEBS Lett 466:333-336). The N-terminus represents an alpha-helix, whereas the C-terminal part has a mixed 3(10)/alpha(R) hydrogen-bond pattern. In the anisotropic micelle environment, the bending angle on Hyp10 (23 degrees) is smaller than that (47 degrees) in isotropic solvents. In the NOESY (Nuclear Overhauser Effect Spectroscopy) spectra, the characteristic attenuation of the peptide signals by 5- and 16-doxylstearate relaxation probes indicates a peripheral mode of the peptaibol binding to the micelle with the N-terminus immersed slightly deeper into micelle interior. Analysis of the surface hydrophobicity reveals that the zervamicin IIB helix is amphiphilic and well suited to formation of a tetrameric transmembrane bundle, according to the barrel-stave mechanism. The results are discussed in a context of voltage-driven peptaibol insertion into membrane.

NMR structure of the channel‐former zervamicin IIB in isotropic solvents

Spatial structure of the membrane channel‐forming hexadecapeptide, zervamicin IIB, was studied by NMR spectroscopy in mixed solvents of different polarity ranging from CDCl3/CD3OH (9:1, v/v) to CD3OH/H2O (1:1, v/v). The results show that in all solvents used the peptide has a very similar structure that is a bent amphiphilic helix with a mean backbone root mean square deviation (rmsd) value of ca. 0.3 Å. Side chains of Trp1, Ile2, Gln3, Ile5 and Thr6 are mobile. The results are discussed in relation to the validity of the obtained structure to serve as a building block of zervamicin IIB ion channels.

MAS NMR structure refinement of uniformly 13C enriched chlorophyll a/water aggregates with 2D dipolar correlation spectroscopy

Abstract 2D MAS dipolar correlation spectroscopy of uniformly 13 C enriched chlorophyll a /water aggregates was performed using broadband RFDR to promote exchange of coherence through homonuclear dipolar couplings. It is shown experimentally that incorporation of TPPI and coherence pathway selection in the RFDR pulse scheme yields virtually pure 2D absorption lineshapes. From a series of 2D correlation spectra collected at two spinning speeds with different mixing times of ∼ 1 and ∼ 10 ms all 13 C resonances of the chlorophyll a molecule were assigned. The spectra with the longer mixing times reveal through-space intermolecular polarization transfer. This demonstrates that structural information can be obtained from uniformly 13 C enriched samples, which paves the way for a full structural characterization of amorphous solids.

Weakly coupled radical pairs in solids: ELDOR in ESE structure studies

Possibilities of the structure determination of radical pairs having fixed geometry with the help of ELDOR in ESE technique are considered. It is demonstrated that one can obtain information on relative orientation of paramagnetic centers in weakly coupled pairs in addition to the energy parameters of the spin Hamiltonian. Appropriate requirements for such experiments are formulated.

MULTIDIMENSIONAL CP-MAS 13C NMR OF UNIFORMLY ENRICHED CHLOROPHYLL

Abstract The progress toward structure refinement of solid-type uniformly 13C enriched ([U-13C]) chlorophyll-containing biological preparations is summarised. Solid state carbon chemical shifts of aggregated [U-13C] bacteriochlorophyll (BChl) c in intact chlorosomes of Chlorobium tepidum and in [U-13C] BChl c aggregates were determined by the application of homonuclear (13C13C) magic angle spinning (MAS) NMR dipolar correlation spectroscopy. It was found that the arrangement of BChl c molecules in the chlorosomes and in the aggregates is highly similar, which provides convincing evidence that self-organisation of the BChl c is the main mechanism to support the structure of the chlorosomes. Additionally, high field 2-D (1H13C) and 3-D (1H13C13C) dipolar correlation spectroscopy was applied to determine solid state proton chemical shifts of aggregated [U-13C] BChl c in intact chlorosomes. From the high-field assignments, evidence is found for the existence of at least two well-defined interstack arrangements.

Alamethicin Topology in Phospholipid Membranes by Oriented Solid-state NMR and EPR Spectroscopies : a Comparison

Alamethicin, a hydrophobic peptide that is considered a paradigm for membrane channel formation, was uniformly labeled with 15N, reconstituted into oriented phosphatidylcholine bilayers at concentrations of 1 or 5 mol %, and investigated by solid-state NMR spectroscopy as a function of temperature. Whereas the peptide adopts a transmembrane alignment in POPC bilayers at all temperatures investigated, it switches from a transmembrane to an in-plane orientation in DPPC membranes when passing the phase transition temperature. This behavior can be explained by an increase in membrane hydrophobic thickness and the resulting hydrophobic mismatch condition. Having established the membrane topology of alamethicin at temperatures above and below the phase transition, ESEEM EPR was used to investigate the water accessibility of alamethicin synthetic analogues carrying the electron spin label TOAC residue at one of positions 1, 8, or 16. Whereas in the transmembrane alignment the labels at positions 8 and 16 are screened from the water phase, this is only the case for the latter position when adopting an orientation parallel to the surface. By comparing the EPR and solid-state NMR data of membrane-associated alamethicin it becomes obvious that the TOAC spin labels and the cryo-temperatures required for EPR spectroscopy have less of an effect on the alamethicin-POPC interactions when compared to DPPC. Finally, at P/L ratios of 1/100, spectral line broadening due to spin-spin interactions and thereby peptide oligomerization within the membrane were detected for transmembrane alamethicin.

Synthesis of Isotopically Labelled L‐Phenylalanine and L‐Tyrosine

A synthetic route to stable-isotope-substituted L-phenylalanine is presented, which allows the introduction of 13C, 15N, and deuterium labels at any position or combination of positions. For labelling of the aromatic ring, a synthetic route to ethyl benzoate (or benzonitrile) has been developed, based on the electrocyclic ring-closure of a 1,6-disubstituted hexatriene system, with in situ aromatization by elimination of one (amino) substituent. Several important (highly isotopically enriched) synthons have been prepared, namely benzonitrile, benzaldehyde, ethyl benzoate, and ethyl diphenyloxyacetate. Labelled L-phenylalanines have been synthesized from both aromatic precursors by initial conversion into sodium phenylpyruvate and subsequent transformation of this intermediate into the L-α-amino acid by an enzymatic reductive amination reaction. In this manner, highly enriched phenylalanines are obtained on the 10-gram scale and with high enantiomeric purities (≥ 99% ee). The method has been validated by the synthesis of [1′-13C]-L-Phe and [2-D]-L-Phe. In addition, two methods are described for the introduction of isotopes into L-tyrosine starting from the isotopically enriched precursors benzonitrile and ethyl benzoate.