8 research outputs found
Oriented Circular Dichroism: A Method to Characterize Membrane-Active Peptides in Oriented Lipid Bilayers
ConspectusThe structures of membrane-bound polypeptides are intimately related
to their functions and may change dramatically with the lipid environment.
Circular dichroism (CD) is a rapid analytical method that requires
relatively low amounts of material and no labeling. Conventional CD
is routinely used to monitor the secondary structure of peptides and
proteins <i>in solution</i>, for example, in the presence
of ligands and other binding partners. In the case of membrane-active
peptides and transmembrane proteins, these measurements can be applied
to, and remain limited to, samples containing detergent micelles or
small sonicated lipid vesicles. Such traditional CD analysis reveals
only secondary structures. With the help of an oriented circular dichroism
(OCD) setup, however, based on the preparation of <i>macroscopically
oriented lipid bilayers</i>, it is possible to address the membrane
alignment of a peptide in addition to its conformation. This approach
has been mostly used for α-helical peptides so far, but other
structural elements are conceivable as well. OCD analysis relies on
Moffitt’s theory, which predicts that the electronic transition
dipole moments of the backbone amide bonds in helical polypeptides
are polarized either parallel or perpendicular to the helix axis.
The interaction of the electric field vector of the circularly polarized
light with these transitions results in an OCD spectrum of a membrane-bound
α-helical peptide, which exhibits a characteristic line shape
and reflects the angle between the helix axis and the bilayer normal.
For parallel alignment of a peptide helix with respect to the membrane
surface (S-state), the corresponding “fingerprint” CD
band around 208 nm will exhibit maximum negative amplitude. If the
helix changes its alignment via an obliquely tilted (T-state) to a
fully inserted transmembrane orientation (I-state), the ellipticity
at 208 nm decreases and the value approaches zero due to the decreased
interactions between the field and the transition dipole.Compared
to conventional CD, OCD data are not only collected in
the biologically relevant environment of a highly hydrated planar
lipid bilayer (whose composition can be varied at will), but in addition
it provides information about the tilt angle of the polypeptide in
the membrane. It is the method of choice for screening numerous different
conditions, such as peptide concentration, lipid composition, membrane
additives, pH, temperature, and sample hydration. All these factors
have been found to affect the peptide alignment in membrane, while
having little or no influence on conformation. In many cases, the
observed realignment could be related to biological action, such as
pore formation by antimicrobial and cell-penetrating peptides, or
to binding events of transmembrane segments of integral membrane proteins.
Likewise, any lipid-induced conversion from α-helix to β-sheeted
conformation is readily picked up by OCD and has been interpreted
in terms of protein instability or amyloid-formation
Transmembrane Polyproline Helix
The third most abundant
polypeptide conformation in nature, the
polyproline-II helix, is a polar, extended secondary structure with
a local organization stabilized by intercarbonyl interactions within
the peptide chain. Here we design a hydrophobic polyproline-II helical
peptide based on an oligomeric octahydroindole-2-carboxylic acid scaffold
and demonstrate its transmembrane alignment in model lipid bilayers
by means of solid-state <sup>19</sup>F NMR. As result, we provide
a first example of a purely artificial transmembrane peptide with
a structural organization that is not based on hydrogen-bonding
Lipid Membrane Association of Myelin Proteins and Peptide Segments Studied by Oriented and Synchrotron Radiation Circular Dichroism Spectroscopy
Myelin-specific
proteins are either integral or peripheral membrane
proteins that, in complex with lipids, constitute a multilayered proteolipid
membrane system, the myelin sheath. The myelin sheath surrounds the
axons of nerves and enables rapid conduction of axonal impulses. Myelin
proteins interact intimately with the lipid bilayer and play crucial
roles in the assembly, function, and stability of the myelin sheath.
Although myelin proteins have been investigated for decades, their
structural properties upon membrane surface binding are still largely
unknown. In this study, we have used simplified model systems consisting
of synthetic peptides and membrane mimics, such as detergent micelles
and/or lipid vesicles, to probe the conformation of peptides using
synchrotron radiation circular dichroism spectroscopy (SRCD). Additionally,
oriented circular dichroism spectroscopy (OCD) was employed to examine
the orientation of myelin peptides in macroscopically aligned lipid
bilayers. Various representative peptides from the myelin basic protein
(MBP), P0, myelin/oligodencrocyte glycoprotein, and connexin32 (cx32)
were studied. A helical peptide from the central immunodominant epitope
of MBP showed a highly tilted orientation with respect to the membrane
surface, whereas the N-terminal cytoplasmic segment of cx32 folded
into a helical structure that was only slightly tilted. The folding
of full-length myelin basic protein was, furthermore, studied in a
bicelle environment. Our results provide information on the conformation
and membrane alignment of important membrane-binding peptides in a
membrane-mimicking environment, giving novel insights into the mechanisms
of membrane binding and stacking by myelin proteins
Characterization of the Immersion Properties of the Peripheral Membrane Anchor of the FATC Domain of the Kinase “Target of Rapamycin” by NMR, Oriented CD Spectroscopy, and MD Simulations
The multidomain ser/thr kinase “target
of rapamycin”
(TOR) centrally controls eukaryotic growth and metabolism. The C-terminal
FATC domain is important for TOR regulation and was suggested to directly
mediate TOR-membrane interactions. Here, we present a detailed characterization
of the membrane immersion properties of the oxidized and reduced yeast
TOR1 FATC domain (2438–2470 = y1fatc). The immersion depth
was characterized by NMR-monitored interaction studies with DPC micelles
containing paramagnetically tagged 5- or 16-doxyl stearic acid (5-/16-SASL)
and by analyzing the paramagnetic relaxation enhancement (PRE) from
Mn<sup>2+</sup> in the solvent. Complementary MD-simulations of micellar
systems in the absence and presence of protein showed that 5-/16-SASL
can move in the micelle and that 16-SASL can bend such that the doxyl
group is close to the headgroup region and not deep in the interior
as commonly assumed. Based on oriented CD (OCD) data, the single α-helix
of oxidized/reduced y1fatc has an angle to the membrane normal of
∼30–60°/ ∼ 35–65° in neutral
and ∼5–35°/∼0–30° in negatively
charged bilayers. The presented experimentally well-founded models
help to better understand how this redox-sensitive peripheral membrane
anchor may be part of a network of protein–protein and protein–membrane
interactions regulating TOR localization at different cellular membranes.
Moreover, the presented work provides a good methodological reference
for the structural characterization of other peripherally membrane
associating proteins
Structure-Based Engineering of a Minimal Porin Reveals Loop-Independent Channel Closure
Porins,
like outer membrane protein G (OmpG) of <i>Escherichia
coli</i>, are ideal templates among ion channels for protein
and chemical engineering because of their robustness and simple architecture.
OmpG shows fast transitions between open and closed states, which
were attributed to loop 6 (L6). As flickering limits single-channel-based
applications, we pruned L6 by either 8 or 12 amino acids. While the
open probabilities of both L6 variants resemble that of native OmpG,
their gating frequencies were reduced by 63 and 81%, respectively.
Using the 3.2 Å structure of the shorter L6 variant in the open
state, we engineered a minimal porin (220 amino acids), where all
remaining extramembranous loops were truncated. Unexpectedly, this
minimized porin still exhibited gating, but it was 5-fold less frequent
than in OmpG. The residual gating of the minimal pore is hence independent
of L6 rearrangements and involves narrowing of the ion conductance
pathway most probably driven by global stretching–flexing deformations
of the membrane-embedded β-barrel
Influence of the Length and Charge on the Activity of α‑Helical Amphipathic Antimicrobial Peptides
Hydrophobic mismatch is important
for pore-forming amphipathic
antimicrobial peptides, as demonstrated recently [Grau-Campistany,
A., et al. (2015) <i>Sci. Rep.</i> <i>5</i>, 9388].
A series of different length peptides have been generated with the
heptameric repeat sequence KIAGKIA, called KIA peptides, and it was
found that only those helices sufficiently long to span the hydrophobic
thickness of the membrane could induce leakage in lipid vesicles;
there was also a clear length dependence of the antimicrobial and
hemolytic activities. For the original KIA sequences, the cationic
charge increased with peptide length. The goal of this work is to
examine whether the charge also has an effect on activity; hence,
we constructed two further series of peptides with a sequence similar
to those of the KIA peptides, but with a constant charge of +7 for
all lengths from 14 to 28 amino acids. For both of these new series,
a clear length dependence similar to that of KIA peptides was observed,
indicating that charge has only a minor influence. Both series also
showed a distinct threshold length for peptides to be active, which
correlates directly with the thickness of the membrane. Among the
longer peptides, the new series showed activities only slightly lower
than those of the original KIA peptides of the same length that had
a higher charge. Shorter peptides, in which Gly was replaced with
Lys, showed activities similar to those of KIA peptides of the same
length, but peptides in which Ile was replaced with Lys lost their
helicity and were less active
Anisotropic Organization and Microscopic Manipulation of Self-Assembling Synthetic Porphyrin Microrods That Mimic Chlorosomes: Bacterial Light-Harvesting Systems
Being able to control in time and space the positioning, orientation, movement, and sense of rotation of nano- to microscale objects is currently an active research area in nanoscience, having diverse nanotechnological applications. In this paper, we demonstrate unprecedented control and maneuvering of rod-shaped or tubular nanostructures with high aspect ratios which are formed by self-assembling synthetic porphyrins. The self-assembly algorithm, encoded by appended chemical-recognition groups on the periphery of these porphyrins, is the same as the one operating for chlorosomal bacteriochlorophylls (BChl's). Chlorosomes, rod-shaped organelles with relatively long-range molecular order, are the most efficient naturally occurring light-harvesting systems., They are used by green photosynthetic bacteria to trap visible and infrared light of minute intensities even at great depths, e.g., 100 m below water surface or in volcanic vents in the absence of solar radiation. In contrast to most other natural light-harvesting systems, the chlorosomal antennae are devoid of a protein scaffold to orient the BChl's; thus, they are an attractive goal for mimicry by synthetic chemists, who are able to engineer more robust chromophores to self-assemble. Functional devices with environmentally friendly chromophoreswhich should be able to act as photosensitizers within hybrid solar cells, leading to high photon-to-current conversion efficiencies even under low illumination conditionshave yet to be fabricated. The orderly manner in which the BChl's and their synthetic counterparts self-assemble imparts strong diamagnetic and optical anisotropies and flow/shear characteristics to their nanostructured assemblies, allowing them to be manipulated by electrical, magnetic, or tribomechanical forces
Anisotropic Organization and Microscopic Manipulation of Self-Assembling Synthetic Porphyrin Microrods That Mimic Chlorosomes: Bacterial Light-Harvesting Systems
Being able to control in time and space the positioning, orientation, movement, and sense of rotation of nano- to microscale objects is currently an active research area in nanoscience, having diverse nanotechnological applications. In this paper, we demonstrate unprecedented control and maneuvering of rod-shaped or tubular nanostructures with high aspect ratios which are formed by self-assembling synthetic porphyrins. The self-assembly algorithm, encoded by appended chemical-recognition groups on the periphery of these porphyrins, is the same as the one operating for chlorosomal bacteriochlorophylls (BChl's). Chlorosomes, rod-shaped organelles with relatively long-range molecular order, are the most efficient naturally occurring light-harvesting systems., They are used by green photosynthetic bacteria to trap visible and infrared light of minute intensities even at great depths, e.g., 100 m below water surface or in volcanic vents in the absence of solar radiation. In contrast to most other natural light-harvesting systems, the chlorosomal antennae are devoid of a protein scaffold to orient the BChl's; thus, they are an attractive goal for mimicry by synthetic chemists, who are able to engineer more robust chromophores to self-assemble. Functional devices with environmentally friendly chromophoreswhich should be able to act as photosensitizers within hybrid solar cells, leading to high photon-to-current conversion efficiencies even under low illumination conditionshave yet to be fabricated. The orderly manner in which the BChl's and their synthetic counterparts self-assemble imparts strong diamagnetic and optical anisotropies and flow/shear characteristics to their nanostructured assemblies, allowing them to be manipulated by electrical, magnetic, or tribomechanical forces