2.1. X-ray diffraction

X-ray crystallography- Among the tools used for structural characterization, X-ray crystallography is the standard method. It enables the elucidation of the atomic structure of three-dimensional LP-lipid complexes and the binding sites of LPs in the cell membrane. The graphic display of atomic structures reveals the binding site location and presence of the bound ligand. X-ray diffraction studies on cell membranes allow the evaluation of the structure of membrane proteins, mechanism of membrane transport proteins and the structure of ion channels (Table 3 ). These aspects of X-ray diffraction applications in membrane science have been reviewed by Yigong Shi [⁶⁸]. Most analytical techniques examine LPs secondary structure, as well as the localization of the individual residues on membranes. Likewise, structural changes in the membrane over the course of peptide interaction is another important criterion that needs to be elucidated. The recent development of X-ray diffraction structure and phase property of fluid membranes with short-range order of lipids in the presence of peptides allows this feature to be examined. Recent advancements in this area transformed X-ray diffraction into a powerful method to elucidate LP-lipid interactions in the hydrated state of the lipid bilayer and spontaneous insertion of LPs into membranes. There are two types of information about LP-lipid interactions that can be obtained from X-ray diffraction techniques. One is data on the position of lipids in the membrane layer (at resolutions up to several angstroms (Å), using the heavy atom labeling technique, and the other is the data given by these techniques related to membrane thickening, thinning and packing during LP-lipid interactions.

Membrane perturbations by AMPs examined by the X-ray diffraction method.

Multiple Wavelength Anomalous Diffraction (MAD)- With the development of Multiple Wavelength Anomalous Diffraction (MAD), considerable improvement has been achieved allowing to decipher the ‘phase shift characteristics’ in biological membranes and model systems [^69,70]. Elucidation of the lipid structure is an important aspect of LPs interaction with membranes, since most of them form pores. With the combined use of MAD and the heavy atom labeling technique, information about the lipid structure of the membrane and position of the AA residues of the LP in the membrane layers are revealed. For example, using thallium as the anomalous scattering atoms, the MAD method has been used to determine the lipid structure of membranes containing gramicidin ion channels [⁶⁹]. Lately, the MAD method was successfully used to solve the lipid structure of the inverted hexagonal phase of a phospholipid with brominated chains. The bromine distribution obtained from the MAD analysis provided the details of the chain packing in the hexagonal unit cell, through the observation of the intensity undulation of the bromine distribution around the unit cell [⁷⁰]. The change in the density profile of the bilayer enables the determination of the location of the LP in the bilayer. For example, White et al. developed bilayer profiling by labeling a specific double bond of the lipid. Using this method, they studied the location and orientation of the membrane-bound peptides [⁷¹].

Vital information about membranes can be retrieved when biomimetic multilamellar lipid membranes supported on solid surfaces are irradiated by X-ray [⁷²]. This method offers a novel and non-destructive approach to investigate the structure of lipid membranes, with and without the presence of membrane-active agents such as LPs. With the aid of modern interface sensitive X-ray scattering techniques, a precise distinction can be made between the normal q _z and parallel q _|| scattering vector component of the membrane bilayer. With this technique, the lateral structure of bilayers from weakly ordered systems can be elucidated. One factor to consider is that LP incorporation into multilamellar structures is achieved under non-spontaneous insertion.

The principal mode of action of antimicrobial LPs is through direct cell membrane interaction, rather than cell lysis. Hydrophobic matching mediated by direct interaction of LPs with cell membranes causes subsequent membrane thinning or thickening and lipid reorientation of the membrane. Hence, the X-ray scattering method should be an apt method to probe LP-lipid interactions in the fluid state of the bilayer. Scattering experiments on lipid films could possibly yield evidence in several ways. For example, the vertical density profile of bilayers r(z) (averaged in the XY plane) and the lateral bilayer irregularities through diffuse scattering, the lateral membrane structure on molecular length scale using Grazing Incidence X-ray Diffraction (GIXD) and the ordering of peptides on the surface of the membrane bilayer through Grazing Incidence Small-Angle X-ray Scattering (GISAXS).

Grazing Incidence X-ray Diffraction (GIXRD)- Studies on the molecular structure of membrane surfaces over the course of LP-lipid interactions is also very important. This can be evaluated using GIXRD [^72,73]. By means of in-plane diffraction of periodically organized lipid films, one can obtain high-resolution information about the membrane surface. In a typical GIXRD measurement an incident X-ray radiation with a 1.5 Å wavelength is set to strike at the air-water interface of the membrane at an incident angle (0.8 α_c), below the critical angle of total reflection (α_c). Otherwise, this would lead to total external reflection, having the refracted waves becoming evanescent waves. Evanescent waves travel below the surface parallel to the interface, with a typical penetration depth of 76 Å. In sufficiently long-range ordered membranes, the ordered structure of monolayers can be diffracted. In the event of LP-lipid interaction, GIXRD would allow the detection of two fundamental factors: firstly, the partial ordering of the peptides and subsequent change in intensity distribution, which can be correlated to pore size, orientation, and conformation [⁷⁴]; secondly, the measurement of area per lipid molecules before and after the introduction of the LP. Using this method, Gidalevitz and coworkers studied the interaction of lipid A (a major component in the outer membrane of gram-negative bacteria) with AMPs such as LL-37, SMAP-29 and D2A22 [⁷⁵]. During a constant pressure experiment, they observed that at higher L/P ratio there was an increase in the area per lipid molecule. Similarly, a study of the interaction between the ovine AMP SMAP-29 and phospholipid monolayers using GIXRD revealed the same proportional increase in the area per lipid [⁷⁶].

According to the study of Huang and co-workers, there was a concentration dependent phase transition occurring at critical peptide-lipid ratio (P/L). They evidenced through circular dichroism (CD) that, in aligned membranes, the transition of α-helical peptides from the parallel state to the inserted state caused a phase transition [^77,78]. The transition from the parallel state to the inserted state of the LP showed a sigmoidal concentration dependence. Furthermore, they established a correlation between this transition and membrane elasticity, as well as other physical parameters such as membrane thickness. During recent years, X-ray diffraction contributed greatly to these studies. First, it was determined that the location of LPs in the bilayer was directly correlated with the changes in the electron density. For example, melittin-MLM in DOPC showed a substantial increase in the PC head group due to its insertion in the bilayer [⁷⁹].

Small-Angle X-ray Scattering (SAXS) and Wide-Angle X-ray Diffraction (WAXD)- Hydrophobic matching and thinning or thickening of the bilayer during interaction with LPs have been studied through Small-Angle X-ray Scattering (SAXS). In-depth analysis of scattering data has been made possible through the use of advanced software and hardware technologies [^80,81]. Due to technological advancements of the third-generation synchrotrons and X-ray detectors, there is a growing demand for SAXS in the structural biology community. Typical SAXS experiments involve recording the scattering at small angles (typically 0.1–10°) and the elastically scattered waves of the X-ray beam impinging on electrons (Fig. 1 ). Unlike other structural techniques, the scattering curve can always be measured without having a well-diffracting crystal, such as the one required for crystallographic analysis. Using background-subtracted SAXD, one can obtain the parameters of the d value that is the sum of membrane thickness (d_B) and thickness of water layer (d_w) [d = d_B + d_w] and the 1D electron density profile calculated from SAXD diffractograms. For instance, Ortiz and coworkers studied the interaction of lichenysin with dipalmitoylphosphatidylcholine (DPPC) membranes via the SAXD method [⁸²]. They revealed that, though the presence of the LP did not alter the lamellar structure organization, the interlamellar repeat distance increased. The electron density profile also revealed that there was an increase in the d value, due to the insertion of lichenysin in the DPPC membrane bilayer. Likewise, the interaction of an antimicrobial surfactant-like peptide containing a cationic head group (Ala)₆(Arg), A₆, with zwitterionic DPPC lipid vesicles was investigated by Hamely and coworkers. SAXD data revealed that the local multilamellar organization of DPPC vesicles was disturbed, possibly due to the swelling effect [⁸³].

Schematic representation of ideal small-angle scattering experiment (i) as a function of the parallel (q_k) and normal (q_z) components of the momentum transfer (q). (ii) in the vicinity of the (specular) q_z axis. In this study, the q_z components (iii) were measured by line scanning under specular conditions (iv). At low q_z, the lipid acyl chain correlation maximum (v) is observed. Lorentzian fits yield the lateral lipid chain distance. The width of the acyl chain peak along qk gives information about the lipid ordering (correlation length); the angular width of the peak corresponds to the acyl chain tilt. In addition, superstructures (vi) and peptide geometries (helix maximum, vii) can be observed in some cases. Figure used from https://doi.org/10.1007/s00249-010-0645-4.

Like SAXS, Wide Angle X-ray Diffraction (WAXD) is another technique giving information about the aliphatic chain lattice, and thus, the bilayer packing at the Å scale; though requiring the presence of crystalline-like ordered phases. In WAXD, the distance from the sample to the detector is shorter, thus allowing for the observation of the diffraction maxima at longer wavelengths [^82,84].

Although the X-ray diffraction method is suitable for pure LP-lipid model system analyses, it has limitations. For example, X-ray diffraction cannot be applied to study the interaction of larger LPs with lipids unless the membrane active segment of the peptide is split (or else the process becomes laborious). Additionally, X-ray diffraction can only give partial information, even for the highly ordered state of the fluid membrane, and it is not possible to quantify the results below a certain threshold. As previously mentioned, in order to get observable scattering peaks, the lipid-peptide ratio should be high. Nevertheless, summing up the results from X-ray diffraction with the one obtained from additional experimental approaches (Molecular Dynamic simulations, solid-state NMR, CD measurements on oriented membranes and other spectroscopic techniques) it is possible to get high-resolution structural information of LP-lipid interactions.