5 research outputs found
Whole-Body Rocking Motion of a Fusion Peptide in Lipid Bilayers from Size-Dispersed <sup>15</sup>N NMR Relaxation
Biological membranes present a highly fluid environment, and integration of proteins within such membranes is itself highly dynamic: proteins diffuse laterally within the plane of the membrane and rotationally about the normal vector of this plane. We demonstrate that whole-body motions of proteins within a lipid bilayer can be determined from NMR <sup>15</sup>N relaxation rates collected for different-sized bicelles. The importance of membrane integration and interaction is particularly acute for proteins and peptides that function on the membrane itself, as is the case for pore-forming and fusion-inducing proteins. For the influenza hemagglutinin fusion peptide, which lies on the surface of membranes and catalyzes the fusion of membranes and vesicles, we found large-amplitude, rigid-body wobbling motions on the nanosecond time scale relative to the lipid bilayer. This behavior complements prior analyses where data were commonly interpreted in terms of a static oblique angle of insertion for the fusion peptide with respect to the membrane. Quantitative disentanglement of the relative motions of two interacting objects by systematic variation of the size of one is applicable to a wide range of systems beyond protein–membrane interactions
Hybrid NMR: A Union of Solution- and Solid-State NMR
Hybrid NMR (hdNMR)
is a powerful new tool that combines the strengths
of solution- and solid-state NMR to measure dipolar, chemical shift,
and quadrupolar tensors in aqueous solution. We introduce the theory
of hdNMR and partially randomly oriented (PRO) crystalline hydrogel
samples. PRO samples produce randomly oriented spectra with characteristic
Pake patterns from the solid state, yet they maintain the high-resolution
dispersion of solution NMR experiments. With new pulse sequences,
we show how hdNMR can be used to measure with high precision the <sup>1</sup>H<sup>α</sup>–<sup>13</sup>C<sup>α</sup> dipolar tensor and carboxylate chemical shift anisotropy tensor
of aspartate. These measurements contain detailed information on the
distribution of electron density, interatomic distances, and the orientation
dependence of molecular motion
Use of Isotropically Tumbling Bicelles to Measure Curvature Induced by Membrane Components
Isotropically
tumbling discoidal bicelles are a useful biophysical
tool for the study of lipids and proteins by NMR, dynamic light scattering,
and small-angle X-ray scattering. Isotropically tumbling bicelles
present a low-curvature central region, typically enriched with DMPC
in the lamellar state, and a highly curved detergent rim, typically
composed of DHPC. In this report, we study the impact of the partitioning
and induced curvature of a few molecules of a foreign lipid on the
bicelle size, structure, and curvature. Previous approaches for studying
curvature have focused on macroscopic and bulk properties of membrane
curvature. In the approach presented here, we show that the conical
shape of the DOPE lipid and the inverted-conical shape of the DPC
lipid induce measurable curvature changes in the bicelle size. Bicelles
with an average of 1.8 molecules of DOPE have marked increases in
the size of bicelles, consistent with negative membrane curvature
in the central region of the bicelle. With bicelle curvature models,
radii of curvature on the order of −100 Å and below are
measured, with a greater degree of curvature observed in the more
pliable L<sub>α</sub> state above the phase-transition temperature
of DMPC. Bicelles with an average of 1.8 molecules of DPC are reduced
in size, consistent with positive membrane curvature in the rim, and
at higher temperatures, DPC is distributed in the central region to
form mixed-micelle structures. We use translational and rotational
diffusion measurements by NMR, size-exclusion chromatography, and
structural models to quantitate changes in bicelle size, curvature,
and lipid dynamics
Structure and Dynamics of Membrane Proteins and Membrane Associated Proteins with Native Bicelles from Eukaryotic Tissues
<i>In vitro</i> studies of protein structure, function,
and dynamics typically preclude the complex range of molecular interactions
found in living tissues. <i>In vivo</i> studies elucidate
these complex relationships, yet they are typically incompatible with
the extensive and controlled biophysical experiments available <i>in vitro</i>. We present an alternative approach by extracting
membranes from eukaryotic tissues to produce native bicelles to capture
the rich and complex molecular environment of <i>in vivo</i> studies while retaining the advantages of <i>in vitro</i> experiments. Native bicelles derived from chicken egg or mouse cerebrum
tissues contain a rich composition of phosphatidylcholine (PC), phosphatidylethanolamine
(PE), phosphatidylglycerol (PG), phosphatidylserine (PS), phosphatidylinositol
(PI), phosphatidic acid (PA), lysolipids, cholesterol, ceramides (CM),
and sphingomyelin (SM). The bicelles also contain source-specific
lipids such as triacylglycerides (TAGs) and sulfatides from egg and
brain tissues, respectively. With the influenza hemagglutinin fusion
peptide (HAfp) and the C-terminal Src homology domain of lymphocyte-specific
protein-tyrosine kinase (lck-cSH2), we show that membrane proteins
and membrane associated proteins reconstituted in native bicelles
produce high-resolution NMR data and probe native protein–lipid
interactions
A Positively Charged Liquid Crystalline Medium for Measuring Residual Dipolar Couplings in Membrane Proteins by NMR
Residual Dipolar Couplings (RDCs)
are integral to the refinement
of membrane protein structures by NMR since they accurately define
the orientation of helices and other structural units. Only a small
set of liquid crystals used for RDC measurements are compatible with
the detergents needed in membrane protein studies. The available detergent-compatible
liquid crystals are negatively charged, thus offering effectively
only one of five orthogonal components of the alignment Saupe matrix.
In this communication, we present a robust liquid crystalline medium
that is positively charged, pinacyanol acetate (PNA), for the determination
of orthogonal sets of RDCs in membrane proteins. This new medium promises
to enhance the accuracy of membrane protein structures and the measurement
of dynamics based on RDCs