Angewandte
Communications
Chemie
NMR Spectroscopy
Delivering Structural Information on the Polar Face of Membrane-
19
Active Peptides: F-NMR Labels with a Cationic Side Chain
Oleg M. Michurin, Sergii Afonin,* Marina Berditsch, Constantin G. Daniliuc, Anne S. Ulrich,
Abstract: Conformationally constrained non-racemizing tri-
fluoromethyl-substituted lysine isosteres [(E)- and (Z)-
TCBLys] with charged side chains are presented as a new
type of 19F-NMR labels for peptide studies. Design of the labels,
their synthesis, incorporation into peptides and experimental
demonstration of their application for solid state NMR studies
of membrane-active peptides are described. A series of
fluorine-labeled analogues of the helical amphipathic antimi-
crobial peptide PGLa(Nle) was obtained, in which different
lysine residues in the original peptide sequence were replaced,
one at a time, by either (E)- or (Z)-TCBLys. Antimicrobial
activities of the synthesized analogues were practically the
same as those of the parent peptide. The structural and
orientational parameters of the helical PGLa(Nle) peptide in
model bilayers, as determined using the novel labels confirmed
and refined the previously known structure. (E)- and (Z)-
TCBLys, as a set of cationic 19F-NMR labels, were shown to
deliver structural information about the charged face of
amphipathic peptides by solid state 19F-NMR, previously
inaccessible by this method.
transport,[3] amyloidogenesis,[4] among others.[5] The structure,
dynamics and alignment of MAPs in lipid bilayers are
important parameters for unraveling the molecular mecha-
nisms of their biological actions.
Only very few of the current techniques are suitable for
structural studies of MAPs in their membrane-bound state
under ambient conditions, because of the inherent complexity
of the membrane systems. This complexity and conforma-
tional plasticity of MAPs make it nearly impossible to
perform X-ray diffraction or electron microscopy studies.
The biophysical methods which use oriented membranes
(fluorescence-,[6] infrared-,[7] and oriented circular dichroism
spectroscopy,[8] colorimetry,[9] interface-sensitive X-ray or
neutron scattering[10] and quartz crystal microbalance[11]
)
provide only low-resolution structural information or suffer
from technical problems (e.g. radiation damage or membrane
dehydration).
As the most appropriate methodology, solid state NMR
spectroscopy (ssNMR) of the peptides labeled by 2H, 15N, 13C,
19F isotopes is used for decades to study MAPs at near-atomic
resolution.[12a] In particular, 19F-ssNMR method has been
successfully used to obtain structural information for more
than a dozen MAPs.[12b–e]
M
embrane-active peptides (MAPs) realize their functions
via interactions with biological membranes and play crucial
roles in many fundamental biological processes, including
innate immune response,[1] membrane fusion,[2] intracellular
Unlike labeling by 2H, 15N or 13C, introduction of
a fluorine-substituted amino acid in place of natural residues,
shown to be useful for qualitative structural assessments,[13]
might significantly change structure and properties of the
labeled peptides.[14] While it might be beneficial for the design
of peptides with improved characteristics for practical appli-
cation, it is completely unacceptable for peptide studies by
19F-ssNMR, where an ideal label should be non-perturbing
and conformationally rigid in order to provide correct
structural constraints by NMR.[12b–d]
[*] O. M. Michurin, Dr. D. S. Radchenko
Enamine Ltd.
vul. Chervonotkatska 78, 02094 Kyiv (Ukraine)
E-mail: Dmitry.Radchenko@mail.enamine.net
Prof. I. V. Komarov
Institute of High Technologies
Taras Shevchenko National University of Kyiv
vul. Volodymyrska 60, 01601 Kyiv (Ukraine)
E-mail: ik214@yahoo.com
Currently known 19F-labels (1–6) are shown in Figure 1;
they were used to substitute non-polar natural amino
acids.[12b–d,15] The only exception is the amino acid 7[16] which
has a polar side chain. To date, no fluorine-substituted amino
acids with charged side chains have ever been described as
labels for 19F-ssNMR studies of MAPs. One of the reasons for
this is the challenging design: a fluorine-containing substitu-
ent will perturb the polar charged side chains much more than
will modify the hydrophobic non-polar aliphatic residues.
Charged amino acid residues are known to be fundamen-
tally important for the structure and functions of numerous
MAPs. For example, interaction of anionic lipid head groups
with the positively charged side chains is a prerequisite for the
MAPs to bind to natural membranes.[17] Another example is
the ladder-like pattern of positively and negatively charged
amino acid residues forming multiple salt bridges, which has
Dr. S. Afonin, Prof. A. S. Ulrich
Institute of Biological Interfaces (IBG-2)
Karlsruhe Institute of Technology (KIT)
POB 3640, 76021 Karlsruhe (Germany)
E-mail: Sergiy.Afonin@kit.edu
Dr. M. Berditsch, Prof. A. S. Ulrich, Dr. D. S. Radchenko
Institute of Organic Chemistry (IOC), KIT
Fritz-Haber-Weg 6, 76131 Karlsruhe (Germany)
Dr. C. G. Daniliuc
Institute of Organic Chemistry
Westfalische Wilhelms-Universitꢀt Mꢁnster
Corrensstrasse 40, 48149 Mꢁnster (Germany)
Supporting information and the ORCID identification number(s) for
Angew. Chem. Int. Ed. 2016, 55, 1 – 6
ꢀ 2016 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
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These are not the final page numbers!