Chemical labeling strategy with (R)- and (S)-trifluoromethylalanine for solid state 19F NMR analysis of peptaibols in membranes

Article abstract of DOI:10.1021/ja9067595

(Figure Presented) Substitution of a single Aib-residue in a peptaibol with (R)- and (S)-trifluoromethylalanine yields two local orientational constraints θ by solid state ¹⁹F NMR. The structure of the membrane-perturbing antibiotic alamethicin

Full text of DOI:10.1021/ja9067595

Published on Web 10/14/2009
Chemical Labeling Strategy with (R)- and (S)-Trifluoromethylalanine for Solid
State 19F NMR Analysis of Peptaibols in Membranes
Daniel Maisch,† Parvesh Wadhwani,‡ Sergii Afonin,‡ Christoph Bo¨ttcher,§ Beate Koksch,§ and
Anne S. Ulrich*,†,‡
Karlsruhe Institute of Technology (KIT), Institut fu¨r Organische Chemie, Fritz-Haber-Weg 6,
76133 Karlsruhe, Germany, KIT, Institut fu¨r Biologische Grenzfla¨chen, P.O. Box 3640, 76021 Karlsruhe, Germany,
and Freie UniVersita¨t Berlin, Institut fu¨r Chemie und Biochemie, Takustr. 3, 14195 Berlin, Germany
Received August 15, 2009; E-mail: anne.ulrich@kit.edu
A chemical 19F-labeling scheme for solid state NMR structure
analysis of peptaibols is introduced here and applied to alamethicin
(ALM). Like other antimicrobial peptides, this amphiphilic molecule
acts by permeabilizing bacterial membranes.1 It can assemble in
lipid bilayers as a putative barrel-stave pore, but its exact backbone
conformation, helix alignment, and oligomeric state are still under
debate.2 Given the high proportion of R-aminoisobutyric acid (Aib)
in peptaibols,3 the correspondingly labeled trifluoromethylalanine
(CF3-Ala) should be ideally suited as a reporter group for 19F NMR.4
Taking advantage of the exceptional sensitivity of fluorine,5 we
determined the conformation, alignment, and dynamics of ALM
in 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC) bilayers.
By introducing the (R)- and (S)-stereoisomers of CF3-Ala as pairs
of separate structural reporters, we gained a maximum number of
NMR constraints from only a few selectively labeled positions.
From the naturally occurring heterogeneous ALM mixture the
sequence of F30/3 (Ac-Aib-Pro-Aib-Ala-Aib-Ala-Gln-Aib-Val-Aib-
Gly-Leu-Aib-Pro-Val-Aib-Aib-Glu-Gln-Phe-ol) was chosen, since
an X-ray structure exists for the crystalline form.6 Liquid state NMR
had confirmed the largely helical conformation,7 which is inter-
rupted by a kink at Pro14. To determine the structure of ALM in
a lipid environment by solid state NMR, we replaced a single Aib
residue in position 5, 10, or 16 by CF3-Ala. This amino acid has
never before been used for this purpose; hence we aimed to examine
it and demonstrate here that it fulfills all criteria of an ideal 19F
NMR label, being highly sensitive, containing a spinning CF3-group
that is attached to the peptide backbone, and being structurally
virtually unperturbing.8 Another novel aspect of the Aib-labeling
strategy is the fact that twice as many unambiguous (see ref 2b)
local NMR parameters can be obtained compared to conventional
amino acids. Namely, for each of the three Aib positions to be
labeled, two epimeric peptides were synthesized with either the
(R)- or (S)-stereoisomer of CF3-Ala, resulting in a total of six
different ALM analogues.
Given that they are separatable, this approach is preferable over
stereoselective synthesis, since the subsequent NMR structure
analysis relies on the use of both epimers. The purity of the peptide
1
fragments was confirmed by HPLC, H and 19F NMR, and mass
spectrometry (see SI). To assign the (R)- and (S)-stereocenters, each
of the six compounds was hydrolyzed and compared by analytical
HPLC with the retention profile of pure H-(R)-CF3-Ala-OH and
H-(S)-CF3-Ala-NH2 on a C18 column with a chiral mobile phase.11
The full-length ALM sequences were then completed by manual
solid phase peptide synthesis and purified by semipreparative HPLC
(see SI).
To examine the structural and functional compatibility of the
CF3-Ala substitutions, circular dichroism (CD) spectra were
measured for ALM F30/3 and the six 19F-labeled analogues under
membrane-mimicking conditions (50% TFE in phosphate buffer,
Figure 1A,B). All line shapes are very similar with a high helical
content of 40-50% according to quantitative deconvolution by
standard algorithms.12 This confirms that a substitution of Aib by
CF3-Ala does not perturb the backbone in either configuration.
Functional tests of all ALM analogues by bacterial growth inhibition
assays gave the same conclusion (see SI).
CF3-Ala was synthesized as reported.9 Its incorporation into the
peptides was challenging, as the CF3-group drastically reduces the
nucleophilicity of the amine, in addition to its obvious steric
hindrance. Since coupling of the subsequent amino acid is slow in
solid phase synthesis,10 three tripeptides were prepared first in
solution [see Supporting Information (SI)] as NR-protected building
blocks: Ala-(CF3-Ala)-Ala, Val-(CF3-Ala)-Gly, and Val-(CF3-Ala)-
Aib.
Figure 1. (A, B) CD spectra of the six 19F-labeled ALM analogues (with
(R)- and (S)-CF3-Aib) and of the wild type F30/3, recorded in 1:1 (v/v)
TFE/buffer. (C) Solid state 19F NMR spectra of the same analogues,
reconstituted in macroscopically aligned DMPC bilayer and measured at
308 K with the membrane normal parallel (0°) to the static magnetic field.
For solid state 19F NMR analysis, all peptides were reconstituted
into macroscopically oriented DMPC bilayers at a molar peptide-
to-lipid (P/L) ratio of 1/10, as previously described for other
peptides.13 The narrow 31P NMR lineshapes of all samples
demonstrated a high quality of lipid alignment (e.g., SI).
The use of racemic N-benzyloxycarbonyl-CF3-Ala yielded
epimeric mixtures for each tripeptide, and the isomers containing
(R)- and (S)-CF3-Ala could be separated by flash chromatography.
The 19F NMR spectra show that all six ALM analogues were
well oriented, since no “powder patterns” were present (Figure 1C).
From the triplet splittings of the CF3-labels the 19F-19F dipolar
† KIT, Institut fu¨r Organische Chemie.
‡ KIT, Institut fu¨r Biologische Grenzfla¨chen.
§ Freie Universita¨t Berlin.
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10.1021/ja9067595 CCC: $40.75  2009 American Chemical Society

C O M M U N I C A T I O N S
couplings could be extracted. Measurements at different sample
inclinations (0° and 90° with respect to magnetic field) show that
the peptides undergo unrestricted rotational diffusion in the liquid
crystalline bilayer on the millisecond time scale, since the splittings
decrease at 90° by a factor of -1/2 due to motional averaging (cf.
previous reports).5b,13,14 The dipolar coupling of each CF3-group
was converted into a local orientational constraint for the corre-
sponding labeled position.5b,15 The resulting six parameters were
used in a grid search to determine the orientation of ALM in terms
of its tilt angle (τ), azimuthal rotational angle (F), and dynamic
parameter Smol, as described in the SI. For an initial assessment,
several different helical conformations were examined as simplified
input models, namely a straight R-helix, a straight 310-helix, and
the crystal structure (PDB-code: 1AMT).
with a kink presumably at Pro14; (ii) the N-terminal helix has a
transmembrane alignment with an 8° tilt angle; and (iii) its mobility
suggests an oligomeric assembly that does not wobble but is neverthe-
less able to diffuse laterally in the bilayer. Given the high sensitivity
of 19F NMR and having demonstrated here the particular utility of
this new Aib-labeling scheme for peptaibols, the inclusion of an
additional pair of CF3-Ala labels in the C-terminus could be used in
the future to complete the picture of the full-length peptide. Thus, it
will be possible to examine the influence of peptide concentration and
lipid composition on the behavior of ALM, specifically concerning
its postulated switch between a monomeric state and an oligomeric
assembly, and to answer the question of whether the kink at Pro14
participates in a conformational change upon activation and pore
formation.
None of these initial model structures gave a self-consistent
solution with an acceptable ꢀ2 minimum. Given the pronounced
and possibly flexible kink at Pro14, we then excluded the two
constraints of position 16. The remaining four constraints on the
N-terminal helical segment (positions 5 and 10) gave an excellent
fit with a low ꢀ2 (Figure 2A). This best-fit solution suggests that
the N-terminal R-helix is oriented with a tilt angle τ of 8° ( 4°,
corresponding to an almost upright transmembrane alignment. The
azimuthal rotation F of nominally 82° ( 30° is intrinsically poorly
defined due to this upright alignment. Other conformations such
as a 310-helix and the crystal structure were also tested, but these
ꢀ2 minima were not satisfactory (see SI). The observed conformation
and alignment of ALM in DMPC (Figure 2B) are thus fully
consistent with previous reports on the orientation of the N-terminal
helix.2a,6,16 The high order parameter (Smol ) 0.99) indicates that
the peptide does not undergo any long-axial wobble in the lipid
bilayer, which is plausible when several monomers are assembled
as an oligomer.17 Indeed, this observation supports the formation
of a barrel-stave pore under the present sample conditions (P/L )
1:10) as has been suggested before.18
Acknowledgment. Financial support from the CFN (E1.2) and
the help of Dr. R. W. Glaser, Dr. U. H. N. Du¨rr (Mathematica
files), and S. Ruden (MIC assays) are gratefully acknowledged.
Supporting Information Available: Full experimental details
regarding materials, methods, synthesis of 19F-labeled Aib, tripeptides
and full ALM analogues, and their characterizations. This material is
available free of charge via the Internet at http://pubs.acs.org.
References
(1) Zasloff, M. Nature 2002, 415, 389–395.
(2) (a) Bak, M.; Bywater, R. P.; Hohwy, M.; Thomsen, J. K.; Adelhorst, K.;
Jakobsen, H. J.; Sørensen, O. W.; Nielsen, N. C. Biophys. J. 2001, 81, 1684–
1698. (b) Bertelsen, K.; Pedersen, J. M.; Rasmussen, B. S.; Skrydstrup, T.;
Nielsen, N. C.; Vosegaard, T. J. Am. Chem. Soc. 2007, 129, 14717–14723.
(c) North, C. L.; Barranger-Mathys, M.; Cafiso, D. S. Biophys. J. 1995, 69,
2392–2397. (d) Salnikov, E. S.; Friedrich, H.; Li, X.; Bertani, P.; Reissmann,
S.; Hertweck, C.; O’Neil, J. D. J.; Raap, J.; Bechinger, B. Biophys. J. 2009,
96, 86–100.
(3) Kirschbaum, J.; Krause, C.; Winzheimer, R. K.; Bru¨ckner, H. J. Pept. Sci.
2003, 9, 799–809.
(4) Koksch, B.; Jakubke, H.-D.; Wenschuh, H.; Dietmeier, K.; Starostin, A.;
Woolley, A.; Dathe, M.; Mu¨ller, G.; Gussmann, M.: Hofmann, H.-J.; Michel,
Th.; Burger, K. In Peptides 1998: Proceedings of the Twenty-Fifths
European Peptide Symposium; Bajusz, S., Hudecz., F., Eds.; Acade´miai
Kiado´: Budapest, 1998; pp 670-671.
(5) (a) Strandberg, E.; Ulrich, A. S. Concepts Magn. Reson. A 2004, 23A, 89–
120. (b) Ulrich, A. S. Prog. Nucl. Magn. Reson. Spectrosc. 2005, 46, 1–21.
(6) Fox, R. O. Jr.; Richards, F. M. Nature 1982, 300, 325–330.
(7) Franklin, J. C.; Ellena, J. F.; Jayasinghe, S.; Kelsh, L. P.; Cafiso, D. S.
Biochemistry 1994, 33, 4036–4045.
(8) (a) Afonin, S.; Glaser, R. W.; Berditchevskaia, M.; Wadhwani, P.; Gu¨hrs, K.-
H.; Mo¨llmann, U.; Perner, A.; Ulrich, A. S. ChemBioChem 2003, 4, 1151–
1163. (b) Mikhailiuk, P. K.; Afonin, S.; Chernega, A. N.; Rusanov, E. B.;
Platonov, M. O.; Dubinina, G. G.; Berditsch, M.; UIrich, A. S.; Komarov,
I. V. Angew. Chem., Int. Ed. 2006, 45, 5659–5661.
(9) Smits, R.; Cadicamo, C. D.; Burger, K.; Koksch, B. Chem. Soc. ReV. 2008,
37, 1727–1739; see references therein and in SI.
(10) (a) Bo¨hm, H. J.; Banner, D.; Bendels, S.; Kansy, M.; Kuhn, B.; Mu¨ller, K.;
Obst-Sander, U.; Stahl, M. ChemBioChem 2004, 5, 637–643. (b) Ja¨ckel, C.;
Koksch, B. Eur. J. Org. Chem. 2005, 21, 4483–4503. (c) Koksch, B.;
Sewald, N.; Hofmann, H.-J.; Burger, K.; Jakubke, H.-D. J. Pept. Sci. 1997,
3, 157–167.
(11) Koksch, B.; Quaedflieg, P. J. L. M.; Michel, T.; Burger, K.; Broxterman,
Q. B.; Schoemaker, H. E. Tetrahedron: Asymmetry 2004, 15, 1401–1407.
(12) (a) Provencher, S. W.; Glöckner, J. Biochemistry 1981, 20, 33–37. (b)
Johnson, W. C. Proteins: Struct., Func., Bioinf. 1999, 35, 307–312.
(13) Wadhwani, P.; Bu¨rck, J.; Strandberg, E.; Mink, C.; Afonin, S.; Ulrich, A. S.
J. Am. Chem. Soc. 2008, 130, 16515–16517.
(14) (a) Afonin, S.; Grage, S. L.; Ieronimo, M.; Wadhwani, P.; Ulrich, A. S. J. Am.
Chem. Soc. 2008, 130, 16512–16514. (b) Grage, S. L.; Ulrich, A. S. J. Magn.
Reson. 1999, 138, 98–106. (c) Cornell, B. A.; Separovic, F.; Baldassi, A. J.;
Smith, R. Biophys. J. 1988, 53, 67–76.
(15) Glaser, R. W.; Sachse, C.; Du¨rr, U. H. N.; Wadhwani, P.; Ulrich, A. S. J.
Magn. Reson. 2004, 168, 153–163.
Figure 2. (A) 19F NMR data yield a F/τ plot with a unique ꢀ2-minimum
for the N-terminal segment (based on the four labels from positions 5 and
10), revealing an R-helical conformation with best-fit values of τ ≈ 8°, F
≈ 82°, and Smol ≈ 1. The two C-terminal constraints from position 16 deviate
from this alignment (when modeled as a straight R-helix or 310-helix),
confirming that the structure is interrupted by a kink. (B) Visualized
alignment of the N-terminal R-helix (in red) of ALM with a tilt angle of τ
≈ 8° relative to the membrane normal in a DMPC bilayer (the kinked
C-terminus is shown gray; drawing created with MOLMOL).19
In summary, three different Aib residues of the peptaibol alamethicin
were substituted with (R)- and (S)-CF3-Ala to obtain six 19F-labeled
analogues by a combination of solution phase and solid phase peptide
synthesis protocols. Having proven that the secondary structure and
antimicrobial function of these ALM analogues remained unperturbed,
the local orientations of the CF3-groups were measured by solid state
19F NMR. Structure calculations yielded a unique and accurate picture
of the N-terminal segment of ALM in DMPC membranes. These
results show per se that (i) its conformation is R-helical (but not 310)
(16) (a) Banerjee, U.; Zidovetzki, R.; Birge, R. R.; Chan, S. I. Biochemistry 1985,
24, 7621–7625. (b) Esposito, G.; Carver, J. A.; Boyd, J.; Campbell, I. D.
Biochemistry 1987, 26, 1043–1050. (c) Yee, A. A.; O’Neil, J. D. J.
Biochemistry 1992, 31, 3135–3143.
(17) Afonin, S.; Du¨rr, U. H. N.; Wadhwani, P.; Salgado, J.; Ulrich, A. S. Top.
Curr. Chem. 2008, 273, 139–154.
(18) Constantin, D.; Brotons, G.; Jarre, A.; Li, C.; Salditt, T. Biophys. J. 2007,
92, 3978–3987.
(19) Koradi, R.; Billeter, M.; Wu¨thrich, K. J. Mol. Graphics 1996, 14, 51–55.
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