4 research outputs found
Detergent-Type Membrane Fragmentation by MSI-78, MSI-367, MSI-594, and MSI-843 Antimicrobial Peptides and Inhibition by Cholesterol: A Solid-State Nuclear Magnetic Resonance Study
Multidrug
resistance against the existing antibiotics is becoming
a global threat, and any potential drug that can be designed using
cationic antimicrobial peptides (AMP) could be an alternate solution
to alleviate this existing problem. The mechanism of action of killing
bacteria by an AMP differs drastically in comparison to that of small
molecule antibiotics. The main target of AMPs is to interact with
the lipid bilayer of the cell membrane and disrupt it to kill bacteria.
Consequently, the modes of membrane interaction that lead to the selectivity
of an AMP are very important to understand. Here, we have used different
membrane compositions, such as negatively charged, zwitterionic, or
mixed large unilamellar vesicles (LUVs), to study the interaction
of four different synthetically designed cationic, linear antimicrobial
peptides: MSI-78 (commercially known as pexiganan), MSI-367, MSI-594,
and MSI-843. Our solid-state nuclear magnetic resonance (NMR) experiments
confirmed that the MSI peptides fragmented LUVs through a detergent-like
carpet mechanism depending on the amino acid sequence of the MSI peptide
and/or the membrane composition of LUVs. Interestingly, the fragmented
lipid aggregates such as SUVs or micelles are sufficiently small to
produce an isotropic peak in the <sup>31</sup>P NMR spectrum. These
fragmented lipid aggregates contain only MSI peptides bestowed with
lipid molecules as confirmed by NMR in conjunction with circular dichroism
spectroscopy. Our results also demonstrate that cholesterol, which
is present only in the eukaryotic cell membrane, inhibits the MSI-induced
fragmentation of LUVs, suggesting that the MSI peptides can discriminate
the bacteria and the eukaryotic cell membranes, and this selectivity
could be used for further development of novel antibiotics
Phosphatidylethanolamine Enhances Amyloid Fiber-Dependent Membrane Fragmentation
The toxicity of amyloid-forming peptides has been hypothesized
to reside in the ability of protein oligomers to interact with and
disrupt the cell membrane. Much of the evidence for this hypothesis
comes from in vitro experiments using model membranes. However, the
accuracy of this approach depends on the ability of the model membrane
to accurately mimic the cell membrane. The effect of membrane composition
has been overlooked in many studies of amyloid toxicity in model systems.
By combining measurements of membrane binding, membrane permeabilization,
and fiber formation, we show that lipids with the phosphatidylethanolamine
(PE) headgroup strongly modulate the membrane disruption induced by
IAPP (islet amyloid polypeptide protein), an amyloidogenic protein
involved in type II diabetes. Our results suggest that PE lipids hamper
the interaction of prefibrillar IAPP with membranes but enhance the
membrane disruption correlated with the growth of fibers on the membrane
surface via a detergent-like mechanism. These findings provide insights
into the mechanism of membrane disruption induced by IAPP, suggesting
a possible role of PE and other amyloids involved in other pathologies
Lipid Composition-Dependent Membrane Fragmentation and Pore-Forming Mechanisms of Membrane Disruption by Pexiganan (MSI-78)
The
potency and selectivity of many antimicrobial peptides (AMPs)
are correlated with their ability to interact with and disrupt the
bacterial cell membrane. <i>In vitro</i> experiments using
model membranes have been used to determine the mechanism of membrane
disruption of AMPs. Because the mechanism of action of an AMP depends
on the ability of the model membrane to accurately mimic the cell
membrane, it is important to understand the effect of membrane composition.
Anionic lipids that are present in the outer membrane of prokaryotes
but are less common in eukaryotic membranes are usually thought to
be key for the bacterial selectivity of AMPs. We show by fluorescence
measurements of peptide-induced membrane permeabilization that the
presence of anionic lipids at high concentrations can actually inhibit
membrane disruption by the AMP MSI-78 (pexiganan), a representative
of a large class of highly cationic AMPs. Paramagnetic quenching studies
suggest MSI-78 is in a surface-associated inactive mode in anionic
sodium dodecyl sulfate micelles but is in a deeply buried and presumably
more active mode in zwitterionic dodecylphosphocholine micelles. Furthermore,
a switch in mechanism occurs with lipid composition. Membrane fragmentation
with MSI-78 can be observed in mixed vesicles containing both anionic
and zwitterionic lipids but not in vesicles composed of a single lipid
of either type. These findings suggest membrane affinity and membrane
permeabilization are not always correlated, and additional effects
that may be more reflective of the actual cellular environment can
be seen as the complexity of the model membranes is increased
Temperature-Resistant Bicelles for Structural Studies by Solid-State NMR Spectroscopy
Three-dimensional structure determination
of membrane proteins
is important to fully understand their biological functions. However,
obtaining a high-resolution structure has been a major challenge mainly
due to the difficulties in retaining the native folding and function
of membrane proteins outside of the cellular membrane environment.
These challenges are acute if the protein contains a large soluble
domain, as it needs bulk water unlike the transmembrane domains of
an integral membrane protein. For structural studies on such proteins
either by nuclear magnetic resonance (NMR) spectroscopy or X-ray crystallography,
bicelles have been demonstrated to be superior to conventional micelles,
yet their temperature restrictions attributed to their thermal instabilities
are a major disadvantage. Here, we report an approach to overcome
this drawback through searching for an optimum combination of bicellar
compositions. We demonstrate that bicelles composed of 1,2-didecanoyl-<i>sn</i>-glycero-3-phosphocholine (DDPC) and 1,2-diheptanoyl-<i>sn</i>-glycero-3-phosphocholin (DHepPC), without utilizing additional
stabilizing chemicals, are quite stable and are resistant to temperature
variations. These <i>temperature-resistant bicelles</i> have
a robust bicellar phase and magnetic alignment over a broad range
of temperatures, between −15 and 80 °C, retain the native
structure of a membrane protein, and increase the sensitivity of solid-state
NMR experiments performed at low temperatures. Advantages of two-dimensional
separated-local field (SLF) solid-state NMR experiments at a low temperature
are demonstrated on magnetically aligned bicelles containing an electron
carrier membrane protein, cytochrome <i>b</i><sub>5</sub>. Morphological information on different DDPC-based bicellar compositions,
varying <i>q</i> ratio/size, and hydration levels obtained
from <sup>31</sup>P NMR experiments in this study is also beneficial
for a variety of biophysical and spectroscopic techniques, including
solution NMR and magic-angle-spinning (MAS) NMR for a wide range of
temperatures