31P NMR Studies of Phospholipids

Andrei V. Filippov*,†,1; Aidar M. Khakimov†; Bulat V. Munavirov†    * Chemistry of Interfaces, Lulea University of Technology, Lulea, Sweden
† Institute of Physics, Kazan Federal University, Kazan, Russian Federation
1 Corresponding author: email address: andrey.filippov@kpfu.ru

1 Introduction

Biological membranes of living cells are formed by lipids, which are predominantly phospholipids (PLs) that contain one phosphorus nucleus per molecule. Because of the amphipathic nature of lipids, they are present in the anisotropic smectic liquid-crystalline phase in biomembranes. Phosphorus nuclear magnetic resonance (NMR) is a convenient technique for studying biomembranes, for reasons that are well known. (i) The 31P isotope of phosphorus has 100% natural abundance, and 31P NMR is less sensitive than 1H but more sensitive than 13C NMR. (ii) 31P nuclei, which are not present in proteins and peptides, provide selectivity for the lipid signal in lipid/protein systems. (iii) Chemical shift anisotropy (CSA) of the 31P nucleus is high, allowing study of lipid structures and variations of lipid headgroups in biomembranes. Application of 31P NMR to lipid biomembrane research has a long history, which began in the early 1970s with the majority of the basic work being done from 1970 to 1980. Nevertheless, interest in the application of this technique to lipid systems and biomembranes studies has not diminished in subsequent years. Figure 1 shows the number of journals published on this topic in the last decade.

31P NMR spectroscopy of PL molecules can be used for two general applications [1]: (1) analysis of PL structures and phases based on anisotropic line shapes and (2) differentiation of individual PL species in mixtures based on their isotropic chemical shift. A number of review papers analyzed results that are directly and indirectly related to 31P NMR spectroscopy of PL systems and biomembranes. The review by Joachim Seelig [2] presents theoretical aspects of 31P NMR and a number of experimental results obtained up until 1977. A modern, more specific 31P NMR review of PLs was published by Schiller and coworkers in 2007 [1], describing many applications of phosphorus NMR in the study of micelles, bicelles, and cells. The reviews by Schiller and Arnold [3] and Lee and coworkers [4] also deal with 31P NMR indirectly associated with biomembranes.

Because of the numerous and diverse results generated by using 31P NMR in lipid systems and biomembranes, we did not aim to present a comprehensive evaluation of all techniques, approaches, and outcomes. Instead, we intended to provide an author's view of this research field that would be of basic and practical interest to those characterizing the chemical–physical properties of lipids in model systems. In particular, the effects of cholesterol (CHOL) near and above the gel–liquid phase-transition temperature are the areas of interest that continue to be explored. Another relatively new subject is liquid–liquid phase separation in two- or multicomponent lipid membranes induced by CHOL or certain peptides, which may result in formation of ordered domains or so-called “rafts” [5,6].

2 Basics of 31P NMR

Phosphorus has 23 known isotopes from 24P to 46P. Nevertheless, only one among them is stable—31P; thus, it is considered a monoisotopic element [7]. 31P has a nuclear spin I = 1/2, which makes it possible to be observed by NMR. The gyromagnetic ratio for 31P is approximately 2.5 times smaller than that of 1H [8]. As a result, in the same magnetic field, 31P is observed at frequencies 2.5 times smaller than that of 1H. For example, when 1H is observed at 400 MHz, 31P is observed at approximately 161 MHz.

The nuclear spin I = 1/2 gives 31P a dipolar nucleus. Dipolar nuclei appear spherical, with a uniform charge distribution over the entire surface [8]. Since the nucleus appears spherical, it disturbs a probing electromagnetic field independent of direction. The result is a strong, sharp NMR signal. Quadrupolar nuclei (whose spin I > 1/2) have a nonspherical charge distribution, and thus give rise to nonspherical electric and magnetic fields. As a result, they present much more complicated spectral shapes—broadened lines, shallow peaks, and difficulties with phasing and integration. It is fortunate that 31P has a dipolar nucleus; however, it is frequently bound to quadrupolar nuclei, which may result in additional complications in the shape of the spectra [8,9]. The magnitude of complications depends on various factors such as the natural abundance of the isotope, the quadrupole moment, and the relative receptivity [8]. Moreover, 31P couples to any other nucleus with a nuclear spin I > 0. Apart from 1H and 13C, 31P nuclei couple to themselves (P–P coupling), fluorine (19F), boron (10B and 11B), and a variety of metals [8]. Coupling to 1H—the most common case for biological systems—can be deactivated using proton...