A particular area of biophysical research in which Raman spectroscopy has great potential is the study of lipid–protein interactions. It is becoming increasingly clear that lipids can modulate membrane-embedded protein function and vice versa. This modulation can take both specific and nonspecific forms, but the underlying framework is defined by the amphiphatic nature of lipids and proteins. Proteins are embedded in the lipid bilayer by virtue of their hydrophilic and hydrophobic moieties expressed in the notion of hydrophobic coupling. Of special interest is the preferential enrichment of aromatic residues (for example, tryptophan [Trp]) in the interface between the hydrophilic aqueous phases and the hydrophobic bilayer core. The reason for this and its relation to hydrophobic coupling is not clear despite intensive research.

In this article, we will discuss the feasibility of using Raman spectroscopic markers for the study of lipid–protein complexes in model systems. Specifically, we will briefly review relevant Raman spectroscopic markers for lipids and proteins and how both kinds of markers can be used in the investigation of membrane-embedded proteins in fully hydrated liposomes. We will present data from a model system consisting of gramicidin incorporated into liposomes of various lipid compositions and discuss the feasibility of using spectroscopic markers for lipid and Trp residue markers.

The amphiphatic character of membrane-spanning proteins and lipid molecules implies that lipids and proteins self-assemble to avoid the high energetic cost of exposing hydrophobic moieties to water (1,2). The interaction — hydrophobic coupling — between lipids and membrane proteins is generally only slightly stronger than the interactions between lipids (3,4), suggesting an important role for unspecific lipid–protein hydrophobic interactions (3). The number of available high-resolution structures of integral membrane spanning proteins is increasing, and there is mounting evidence for regulation of membrane proteins by the host bilayer membrane through the hydrophobic coupling between membrane and proteins (4,5). This not only raises specific questions as to how membrane-spanning proteins interact with the lipid bilayer, but also opens the possibility to design biomimetic membranes based upon integral membrane proteins incorporated into encapsulated–supported systems for sensor and separation purposes (6).

One of the specific questions relates to a peculiar feature of integral membrane proteins: namely, a preference for aromatic (for example, Trp) residues located in the interfacial region between the hydrophobic core and the hydrophilic surface of the membrane. This has led to the notion of anchoring residues, in which the anchoring effect is thought to arise from the amphiphatic nature of interfacial aromatic residues (7,8). Despite considerable experimental progress using nuclear magnetic resonance (NMR), electron spin resonance (ESR), and molecular modeling (9–12) in the investigation of the effect of anchoring aromatic (and polar) residues, many issues remain unresolved.

Raman spectroscopy has immediate appeal in the study of lipid–protein interactions because it is a noninvasive and nondestructive technique, and the excitation wavelength can be chosen to be below the absorption frequency of water (13). However, Raman spectroscopy of biomembranes in aqueous solution is a technical challenge. The inherent weakness of Raman scattering and the low concentration of solvated samples makes the collection of a spectrum with a high signal-to-noise ratio difficult. But with increased sensitivity and recent developments in instrumentation, Raman spectroscopy is experiencing a revival in many research areas, including the study of lipids and lipid–protein complexes (14–19).

Here, we first discuss the technical challenges and relevant Raman spectroscopic markers for investigating the role of lipid–protein interactions in fully hydrated protein-vesicle samples. In particular, we present markers relevant for lipid molecular conformations and markers for the conformation of anchoring aromatic residues. Then we present a model system and give a few examples of how Raman markers appear in spectra of this system.

Raman Spectroscopy on Lipid–Protein Complexes in Aqueous Solution

Liposomes present a versatile tool for investigating lipid–protein interactions where one can fully control the lipid composition, protein content, and aqueous phases on both sides of the liposomal membrane. However, the use of protein-incorporated liposomes in Raman spectroscopy presents challenges compared to Raman spectroscopy on solid (powder) samples (19). Longer integration time is needed for aqueous solution samples and the intensity peaks appear broader (20). Also, containers used to avoid evaporation and contamination cause problems like loss of intensity and interferences, so windowless, liquid captive cells (21) are sometimes preferred (20). Raman spectroscopy on biochemical compounds isolated from living systems (and in particular, aromatic residues in membrane proteins) is made difficult due to autofluorescence, which can make the Raman bands "barely detectable" (20).

A longer excitation wavelength can alleviate the problem because fluorescence is frequency dependent. Excitation in the near-infrared (NIR) spectrum provides maximum intensity for a fluorescence-free spectrum and is therefore usually recommended for the study of biological samples. Comparing Raman excitations between 406 and 830 nm on tissues, the best defined lipid features are obtained at 782 and 830 nm (that is, with an excitation in the very near IR) (22). In fact, 785 nm seems to be the best suited excitation wavelength for the identification of a wide range of biomarkers (23). The excitation wavelength imposes constraints, such as the power available, the spot size, the exposure time, grating, and charge-coupled device (CCD) used. The comparison of mapping at 532 nm versus 785 nm illustrates the difference between actual collections at those two frequencies and the actual superiority of excitation in the NIR for this type of application (24).