There are various biophysical techniques used for determining both the secondary and tertiary structures of membrane peptides, in order to understand the mechanism of their uptake, as well as their actions on the membranes.
The methods are mainly used to investigate the peptide’s orientation relative to their thermodynamic properties, and their binding abilities. Currently, it has been reported that there are 313 AMP structures present in the Protein Data Bank – PDB. In this piece, we will explore the various methodologies that were applied in the determination of these, and other structures that have been established so far.
Diffraction – X-ray and Neutron Scattering Technique
The application of x-ray diffraction has been the main method for determining the 3D structure of proteins. This method is based on the diffraction of x-rays whenever they land on a surface, which, in this case, is the protein crystal.
The x-rays are directed towards a protein crystal, and then the resulting data is processed to reveal the structure of the molecules under consideration. While some anti-microbial peptides and cell-penetrating peptides crystal structures, are already present in the protein data bank, it should be noted that a good number of the peptides have very small and flexible sizes, and as such, they usually resist crystallization.
Also, it is never easy to correctly determine the crystal structures of antimicrobial and cell-penetrating peptides together, with biological membranes.
The main idea behind neutron diffraction, is similar to that of x-ray diffraction, only that in neutron diffraction, a beam of cold or thermal neutrons is used in the determination of diffraction patterns, which are then used to identify the structure, without the need for further radiation damage.
For example, the exchange between water molecules and the molecules of deuterium oxide, may be used to obtain vital data on the content of water in the peptide-membrane structure. Neutrons that show a high depth of penetration are ideal for studying the structures of biological membranes, however, the nuclear reactor necessary for producing neutrons, is rather very costly.
Nuclear Magnetic Resonance Spectroscopy
Nuclear magnetic resonance spectroscopy is currently one of the most preferred methods for analyzing membrane dynamics, with a peptide. This technique uses local magnetic fields that are produced by radio waves emanating from the atomic nuclei of the sample under study.
The resonance frequency of the intramolecular magnetic field around an atom in a molecule, usually changes, which in turn, leads to a more detailed analysis of the electronic structure of a molecule, as well as its individual functional group. Currently, most of the structures of the cell-penetrating peptides, as well as the antimicrobial peptides present in the protein data bank, were obtained through solutions of nuclear magnetic resonance spectroscopy – NMR.
While it is possible to get information on the structure of the solutions of the peptide, as well as their flexible region with the use of NMR spectroscopy, the use of solid-state spectroscopy has been used to obtain information regarding the structure, as well as the orientation of the membrane peptides. The use of aqueous buffers in solution-NMR, however, has not been very effective due to the low solubility of hydrophobic or amphipathic peptides. Due to this, tetrafluoroethylene (TFE) and water mixtures or detergent micelles, are usually used as model systems in the examination of membrane protein/peptides.
The choice of the membrane model under consideration may have a significant impact on the secondary structure of the peptide. This implies that the peptide may take a structure that is completely different than the one present in the lipid bilayer.
The mixture used may also have a significant impact on the curvature of the membrane, and consequently, the use of planar lipid bilayers may be more appealing under the circumstances. With solid-state NMR-spectroscopy, it is possible to get high-resolution structures of the peptides, but in disordered phospholipid bilayers.
It can also be used to give high resolutions for the various changes that might be happening in the membranes following the interactions with the lipids. As such, it is effective in providing crucial information about the dynamics, topology, structure, and aggregation of peptides, including the conformational, as well as the supramolecular characteristics of the lipid-peptide assembly.
Some of the most outstanding features of solid-state NMR spectra are their contribution to the chemical shifts, the quadrupolar, and the dipolar interactions. Phosphorus and deuterium are some of the most frequently used NMR nuclei for examining lipid-peptide systems.
Phosphorous is mostly used in studying the interactions taking place between the lipid and the peptide head groups. Deuterium, on the other hand, is mostly used in getting information relating to the lipid-tail dynamics, as well as lipid-tail orientation.
Circular Dichroism Spectroscopy
Circular Dichroism Spectroscopy is when the difference in the absorption rates between the left-handed and the right-handed circularly polarized light, is used in absorption spectroscopy. Circular dichroism – CD, may be useful in studying the secondary structures of peptides in solutions. The primary reason for this is because random coils, and alpha-helix and beta-sheet generally have easily recognizable CD spectra.
Though it is possible for amphiphilic peptides to form random coil structures when they are in solution, they usually portray alpha-helical and beta-sheet structures, whenever they bind to the membrane. With conventional CD, it is possible to obtain the information of a peptide in solution, as well as any structural changes that might be experienced concerning the peptide, upon binding.
The information obtained from the structural changes upon binding, can be effective in estimating the ratio of the free peptides to the bound peptides. However, it is not possible to get this information with the use of traditional CD, as well as the orientation of the peptide relative to the membrane.
Consequently, the application of this approach is limited to working on samples in detergent micelles or liposomes, or samples that have small sonicated lipid vesicles. There is a variation of CD known as Oriented Circular Dichroism – OCD, which heavily relies on the Moffitt theory for structural analysis of membrane peptides. According to this theory, one of the peptides' transitions in a helix, is polarized parallel to the helical axis.
This variation of OCD is normally done on microscopically oriented samples, because this is the best way to deal with issues of membrane alignment of a peptide, as well as its conformation. Yet, this structural analysis method is primarily used for the study of alpha-helical peptides.
One of the most powerful tools for characterizing peptide-membrane interactions, and associated secondary processes, such as peptide-induced lipid transition, membrane permeabilization, and peptide aggregation, at the membrane surface, is known as Isothermal titration calorimetry – ITC. Assuming that the binding heat is constant, it is possible to find out the number of bound ligands from the total amount of heat change.
The enthalpic and entropic changes, as well as the binding constant of the binding process, can always be obtained, by simply calculating the ITC. One of the top benefits of ITC, is that it is possible to use it together with unmodified, native forms of molecules, and as such, using it doesn’t come with the introduction of any artifacts.
Also, ITC is usually one of the most preferred approaches for analyzing cell-penetrating peptides, which may already be coupled with larger molecules. The reason for this, is that CD and NMR spectra of such large molecules, are never easy to analyze. Unlike NMR, the use of ITC doesn’t require you to have a large number of samples.
Differential scanning calorimetry – DSC, is another valuable tool that can be used to obtain information about the extent of the interaction of peptides, with certain lipid components, such as the specificities of peptides for various target lipids. In essence, the primary role of DSC, is to monitor any changes that may be happening, as the phase transition temperatures, as well as the thermodynamics of lipid phase transitions. The results are then used in the analysis of the interaction mechanisms of peptides with lipids, because the lipid membranes are usually disrupted or disorganized.
Atomic Force Microscopy
Atomic Force Microscopy – AFM, is one of the most preferred structural analysis methods for getting information about the restructuring and destabilization of the membranes in the presence of peptides through obtaining the molecular-scale topographical details at the surfaces. The images obtained through AFM, detail subtle changes in the cell heights, as well as the surface roughness after treatment is completed with a peptide.
Sadly, though, this is limited only to the study of membrane mimic systems, which usually has insufficient topographical contrast. This is always the case with peptides that have been inserted into a membrane. Additionally, because AFM has the ability to identify the structures of membrane peptides, based on the size, as well as the shape only, it profoundly lacks the much-desired chemical specificity for these kinds of analyses.
The other notable methods used for the structural analysis of membrane peptides, include techniques such as Fourier Transform Infrared Spectroscopy, and Dynamic Light Scattering.
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