Plasma
membranes are assumed to be highly compartmentalized, which
is believed to be important for the membrane protein functionality.
The liquid ordered-disordered phase segregation in the membranes results
in nanoscale liquid-ordered assemblieslipid rafts. Double
electron–electron resonance spectroscopy (DEER, also known
as PELDOR) is sensitive to spin–spin dipolar interactions between
spin labels at the nanoscale range of distances. Here, DEER is applied
to spin-labeled cholestane, 3β-doxyl-5α-cholestane (DChl),
diluted in bilayers composed of an equimolar mixture of dioleoyl-glycero-phosphocholine
(DOPC) and dipalmitoyl-glycero-phosphocholine (DPPC) phospholipids,
with cholesterol (Chol) added. The DEER data allowed us to detect
clustering of the DChl molecules. Their lateral distribution in the
clusters in the absence of Chol was found to be random, while in the
presence of Chol it became quasi-regular. DEER time traces are fairly
well simulated within a simple square superlattice model. For the
20 mol % Chol content, for which at physiological temperatures, the
lipid rafts are formed, the found superlattice parameter was 3.7 nm.
Assuming that lipid rafts are captioned upon shock freezing at the
temperature of investigation (80 K), the found regularity of DChl
lateral distribution was interpreted by raft substructuring, with
the DChl molecules embedded between the substructures.
In glassy substances and biological media, dynamical transitions are observed in neutron scattering that manifests itself as deviations of the translational mean-squared displacement, 〈x〉, of hydrogen atoms from harmonic dynamics. In biological media, the deviation occurs at two temperature intervals, at ∼100-150 K and at ∼170-230 K, and it is attributed to the motion of methyl groups in the former case and to the transition from harmonic to anharmonic or diffusive motions in the latter case. In this work, electron spin echo (ESE) spectroscopy-a pulsed version of electron paramagnetic resonance-is applied to study the spin relaxation of nitroxide spin probes and labels introduced in molecular glass former o-terphenyl and in protein lysozyme. The anisotropic contribution to the rate of the two-pulse ESE decay, ΔW, is induced by spin relaxation appearing because of restricted orientational stochastic molecular motion; it is proportional to 〈α〉τ, where 〈α〉 is the mean-squared angle of reorientation of the nitroxide molecule around the equilibrium position and τ is the correlation time of reorientation. The ESE time window allows us to study motions with τ < 10 s. For glassy o-terphenyl, the 〈α〉τ temperature dependence shows a transition near 240 K, which is in agreement with the literature data on 〈x〉. For spin probes of essentially different size, the obtained data were found to be close, which evidences that motion is cooperative, involving a nanocluster of several neighboring molecules. For the dry lysozyme, the 〈α〉τ values below 260 K were found to linearly depend on the temperature in the same way as it was observed in neutron scattering for 〈x〉. As spin relaxation is influenced only by stochastic motion, the harmonic motions seen in ESE must be overdamped. In the hydrated lysozyme, ESE data show transitions near 130 K for all nitroxides, near 160 K for the probe located in the hydration layer, and near 180 K for the label in the protein interior. For this system, the two latter transitions are not observed in neutron scattering. The ESE-detected transitions are suggested to be related with water dynamics in the nearest hydration shell: with water glass transition near 130 K and with the onset of overall water molecular reorientations near 180 K; the disagreement with neutron scattering is ascribed to the larger time window for ESE-detected motions.
Free
fatty acids play various important roles in biological membranes.
Double electron–electron resonance spectroscopy (DEER, also
known as PELDOR) of spin-labeled biomolecules is capable of studying
magnetic dipole–dipole (d-d) interactions between spin labels
at the nanoscale range of distances. Here, DEER is applied to study
intermolecular d-d interactions between doxyl-spin-labeled stearic
acids (DSA) in gel-phase phospholipid bilayers composed either of
an equimolecular mixture of 1,2-dipalmitoyl-sn-glycero-3-phosphocholine
and 1,2-dioleoyl-sn-glycero-3-phosphocholine or of
1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine.
DEER data obtained for different DSA concentrations showed that DSA
molecules at their concentration in the bilayer χ larger than
0.5 mol % are assembled into lateral lipid-mediated clusters, with
a characteristic intermolecular distance of 2 nm. Some evidences were
obtained indicating that clusters may consist of “subclusters”,
alternatively appearing in two opposite leaflets. Conventional electron
paramagnetic resonance (EPR) spectra for the gel-phase bilayers showed
that for χ larger than 2 mol % the molecules in the clusters
stick together, forming oligomers. Room-temperature EPR spectra for
the liquid-crystalline phase were found to change noticeably for χ
larger than 0.5 mol %, which may indicate the clustering in a liquid-crystalline
phase similar to that observed by DEER in the gel phase.
Double electron−electron resonance (DEER, also known as PELDOR) and circular dichroism (CD) spectroscopies were explored for the purpose of studying the specificity of the conformation of peptides induced by their assembly into a selfrecognizing system. The E and K peptides are known to form a coiledcoil heterodimer. Two paramagnetic TOAC α-amino acid residues were incorporated into each of the peptides (denoted as K** and E**), and a three-dimensional structural investigation in the presence or absence of their unlabeled counterparts E and K was performed. The TOAC spin-labels, replacing two Ala residues in each compound, are covalently and quasi-rigidly connected to the peptide backbone. They are known not to disturb the native structure, so that any conformational change can easily be monitored and assigned. DEER spectroscopy enables the measurement of the intramolecular electron spin−spin distance distribution between the two TOAC labels, within a length range of 1.5−8 nm. This method allows the individual conformational changes for the K**, K**/E, E**, and E**/K molecules to be investigated in glassy frozen solutions. Our data reveal that the conformations of the E** and K** peptides are strongly influenced by the presence of their counterparts. The results are discussed with those from CD spectroscopy and with reference to the already reported nuclear magnetic resonance data. We conclude that the combined DEER/TOAC approach allows us to obtain accurate and reliable information about the conformation of the peptides before and after their assembly into coiled-coil heterodimers. Applications of this induced fit method to other two-component, but more complex, systems, like a receptor and antagonists, a receptor and a hormone, and an enzyme and a ligand, are discussed.
scite is a Brooklyn-based organization that helps researchers better discover and understand research articles through Smart Citations–citations that display the context of the citation and describe whether the article provides supporting or contrasting evidence. scite is used by students and researchers from around the world and is funded in part by the National Science Foundation and the National Institute on Drug Abuse of the National Institutes of Health.