Techniques
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Techniques
Neutron Diffraction and Reflectometry. How can neutron diffraction (ND) and neutron reflectometry (NR) be used to probe molecular interactions of peptides and proteins in lipid bilayers? Neutrons have a scattering potential that only depends on the nuclear composition and interact quite differently with deuterium than with hydrogen atoms. The difference is revealed by the neutron scattering length (b) that underlies neutron scattering. Because b is positive for deuterium (b2HH = 6.67 x10-5 Å) and negative for hydrogen (bH=-3.64 x10-5 Å) specific atoms or group of atoms can be “highlighted” by means of specific deuteration. The contrast enhancement happens with virtually no effect on the behavior of the studied molecule (isomorphous). Comparison of neutron scattering data from selectively deuterated macromolecular assemblies with those from protonated assemblies under identical experimental conditions allows one to surmise the structure, shapes, and spatial relationships of the assemblies in membranes. The neutron techniques can provide unique details on structural organization and interactions of membranes with peptides and proteins, not available by other techniques, such as peptide conformation, tilt, position and bilayer structure perturbations.
ND measurements from assemblies of oriented lipid multilayers with incorporated peptides uses principles of Bragg diffraction to determine the spatial arrangement of atoms in the measured molecular structure. Most commonly, out-of-plane diffraction from planar bilayer assemblies is used to probe the distribution of atoms in the bilayer on an axis (z) perpendicular to the bilayer plane. Bragg peak intensities are used to calculate, by Fourier inversion, the scattering length density (SLD) distribution of the components in the membrane, in a model-independent manner The use of deuteration is particularly important to highlight structural details and enhance structural resolution for thermally disordered systems such as the lipid bilayer. ND measurements can be performed on either a neutron reflectometer, or a small angle scattering instruments.
NR technique can also be used to characterize molecularly organized lipid layers on the sub-nanometer level. A great advantage of this technique derives from the possibility to study the system under bulk solvent conditions and exchange of materials and buffer in situ. For NR, unlike ND, the SLD profiles of the layers are calculated, indirectly, by assuming reasonable models for the spatial layered distribution of components. Solid-supported lipid monolayers or bilayers suitable for NR measurements can be obtained by various techniques. One particularly useful assembly system used extensively in the co-Is’ labs is the tethered bilayer membrane (tBLM). It consists of a gold coated planar substrates on which single bilayer membranes can be assembled via a polymeric tether that separate the membrane from the solid surface via a sub-bilayer water reservoir. The tBLM provides a robust membrane platform system for investigations by NR and by other surface-sensitive techniques such as SPR and EIS.
Neutron instrumentation
The NCNR currently offers three reflectometers (NG7, PBR and MAGIk) and a suite of small angle scattering instruments that are suitable for the described measurements. The Advanced Neutron Reflectometer/Diffractometer (AND/R), currently MAGIk, is one of the most powerful instruments of this kind worldwide and offers excellent performance for both reflectometry and diffraction. Additionally, a white-beam reflectometer (CANDOR) is being developed, in vision of reducing measurement time (by a factor of ~ 10), improving data statistics and resolution.
Surface sensitive techniques
Lipid bilayers can be described broadly by using a simple macroscopic model. The bilayer can be viewed as a thin insulating slab (the hydrocarbon core) separating two electrically conducting layers (headgroups and salt solution), characterized by a capacitance (Cm) and a resistance (Rm) (Figure). Because the bilayer is a good insulator it can sustain charge across it for a significant period of time. Ion channels or other transporters can affect how long the membrane can hold charge, because they modify the resistance Rm to the ion passage. The ability to store charge is a key to energy storage and conversion by biological membranes. These properties have been utilized to develop sensitive analytical techniques to address permeation through membranes. Patch clamp techniques have become the workhorse of ion channel research, providing important insight into the gating mechanisms and actions (e.g. blockage, inhibition, excitation) of endogenous and synthetic molecules of pharmacological relevance.
Based on similar principles, surface-sensitive techniques applied to bilayer supported on electrodes (e.g. Electrical Impedance Spectroscopy (EIS)) have gained popularity due to their versatility and sensitivity to low amounts of analyte. The EIS data is most useful when collected concomitant with measurements of the binding kinetics of the peptides as they partition from the aqueous phase above into the bilayer membrane, using Surface Plasmon Resonance (SPR) methods. Thus, protein accumulation to the membrane surface (binding kinetics) and the onset of permeabilization through transmembrane protein pores or channels can be corroborated. Macroscopic changes in the membrane properties can be determined as membrane-active molecules are titrated into the measurement chamber.
Scientists at IBBR have developed an integrated EIS/SPR instrument suitable for biological applications, based on a robust and reproducible tethered bilayer platform. To date, only proteins that spontaneously form channels though membranes upon partitioning from the aqueous phase into the membrane have been investigated. However, efforts are made toward immobilization and integration of transmembrane proteins into these systems for interrogation of their response to effector molecules and buffer conditions. The EIS/SPR combined system can be customized to fit various biological applications, from minute detections of analytes to larger scale setups for concomitant structural measurements of the single bilayer by NR. This customized apparatus and the measurement techniques developed by scientists at IBBR surpass the capabilities achieved with commercial devices.