Effects of the osmolyte trimethylamine-N-oxide on conformation, self-association, and two-dimensional crystallization of myelin basic protein

Abstract

The osmolyte trimethylamine-N-oxide (TMAO) is a naturally in vivo occurring “chemical chaperone” that has been shown to stabilise the folding of numerous proteins. Myelin basic protein (MBP) is a molecule that has not yet been suitably crystallized either in three dimensions for X-ray crystallography or in two dimensions for electron crystallography. Here, we describe lipid monolayer crystallization experiments of two species of recombinant murine MBP in the presence of TMAO. One protein was unmodified, whereas the other contained six Arg/Lys → Gln substitutions to mimic the effects of deimination (i.e., the enzymatic modification of Arg to citrulline), which reduces the net positive charge. Planar arrays of both proteins were formed on binary lipid monolayers containing a nickel-chelating lipid and a phosphoinositide. In the presence of TMAO, the diffraction spots of these arrays became sharper and more distinct than in its absence, indicating some improvement of crystallinity. The osmolyte also induced the formation of epitaxial growth of protein arrays, especially with the mutant protein. However, none of these assemblies was sufficiently ordered to extract high-resolution structural information. Circular dichroic spectroscopy showed that MBP gained no increase in ordered secondary structure in the presence of TMAO in bulk solution, whereas it did in the presence of lipids. Dynamic light-scattering experiments confirmed that the MBP preparations were monomodal under the optimal crystallization conditions determined by electron microscopy trials. The salt and osmolyte concentrations used were shown to result in a largely unassociated population of MBP. The amino acid composition of MBP overwhelmingly favours a disordered state, and a neural-network-based scheme predicted large segments that would be unlikely to adopt a regular conformation. Thus, this protein has an inherently disordered nature, which mitigates strongly against its crystallization for high-resolution structure determination.

Introduction

The tertiary structure of myelin basic protein (MBP)¹ and its precise organisation within normal compacted and disrupted myelin multilayers are not yet known but are essential to understanding the mechanisms of development of autoimmunity in multiple sclerosis (Moscarello, 1997; Wood and Moscarello, 1997; Zand et al., 1998). We have constructed a three-dimensional (3D) atomic model of the 18.5 kDa isoform of human MBP (hMBP) on the basis of transmission electron microscopical (TEM) and other published data (Beniac et al., 1997, Beniac et al., 2000; Ridsdale et al., 1997). Recently, we described a recombinant form of the 18.5-kDa isoform of murine MBP (rmMBP) expressed with a carboxyl-terminal hexahistidine tag (Bates et al., 2000). Incubation of this rmMBP with lipid monolayers containing a nickel-chelating lipid (Kubalek et al., 1994) resulted in the formation of packed fibers with a spacing of 4.8 nm between fibers and a longitudinal repeat distance of roughly 4 nm (Bates et al., 2000; Ishiyama, 2000; Ishiyama et al., 2001, Ishiyama et al., 2002). Upon in vitro deimination of the rmMBP preparation to an average of 9 citrulline residues per molecule of protein, closely packed arrangements of particles of 4 nm in diameter were observed, indicating an altered interaction of the protein with lipid and with itself (Ishiyama et al., 2000, Ishiyama et al., 2001). At high salt concentrations, the deiminated protein formed crystalline arrays, the images of which showed periodicity to one diffraction order, corresponding to a spatial resolution of about 4 nm.

The technique of two-dimensional (2D) crystallization and TEM of proteins on lipid monolayers is one that is being increasingly applied to structure determination of proteins, especially membrane-associated ones (Ellis and Hebert, 2001; Hasler et al., 1998; Lévy et al., 1999, Lévy et al., 2001; Mosser, 2001; Walz and Grigorieff, 1998). Since MBP is considered to have a primary role in maintaining the stability of the central nervous system (CNS) myelin sheath by holding together the apposing surfaces of the oligodendrocyte membrane (Martenson, 1980; Moscarello, 1997; Smith, 1992; Staugaitis et al., 1996), this approach has much to commend it. It proffers great versatility in experimental design, especially the ability to mimic the protein’s native lipid environment in the myelin sheath, under which conditions the protein gains ordered secondary structure (Martenson, 1980; Polverini et al., 1999; Smith, 1992). The factors that one can vary in this experimental design include the kind and proportion of the diluting (or “filler” or “helper”) lipid, the kind of buffer, the pH, the ionic strength, and the temperature and time of incubation. In our previous work, we sampled these parameters systematically (Ishiyama, 2000). Unfortunately, we have not been able to obtain planar crystals that diffract even to moderate resolution.

On the basis of its conformational behaviour as assessed spectroscopically (e.g., Polverini et al., 2001, and references therein), MBP can be classified as a “natively unfolded” or “intrinsically unstructured” protein (Baskakov et al., 1999; Dyson and Wright, 2002; Ulrich et al., 2000; Uversky et al., 2000, Uversky et al., 2001; Weinreb et al., 1996; Wright and Dyson, 1999). These proteins have a low overall hydrophobicity and a high net charge, exist in numerous conformations depending on the environment, and, like MBP (Kursula, 2001; Sedzik and Kirschner, 1992), have been difficult or impossible to crystallize for X-ray diffraction. Compounds called osmolytes are used by some organisms to stabilise their macromolecules’ structures under conditions of denaturing stress (Baskakov and Bolen, 1998; Bolen and Baskakov, 2001; Gonnelli and Strambini, 2001; Qu et al., 1998). For example, cartilaginous fish offset the protein-perturbing effects of high intracellular urea concentrations or high hydrostatic pressures by accumulating structure-stabilising methylamines (Yancey and Siebenaller, 1999; Yancey and Somero, 1979; Yancey et al., 1982, Yancey et al., 2001).

One such osmolyte, trimethylamine-N-oxide (TMAO), has been used in many recent studies as a “chemical chaperone” to induce and/or stabilise the folding of numerous proteins: a fragment of the human glucocorticoid receptor in which the α-helical content was significantly increased (Baskakov et al., 1999; Kumar et al., 2001), human apolipoprotein C-1 (Gursky, 1999), the amyloid-β(1-40) peptide (Yang et al., 1999), ribonuclease A (Palmer et al., 2000), tau (Eidenmuller et al., 2000; Smith et al., 2000), ribonuclease S (Ratnaparkhi and Varadarajan, 2001), α-synuclein (Uversky et al., 2001), and other works not cited here. Thus, this compound is a natural in vivo and in vitro inducer of ordered secondary and tertiary structure, in contrast to commonly used organic solvents, such as trifluoroethanol, which induce potentially spurious α-helicity in any protein (Main and Jackson, 1999), including MBP (Bates et al., 2000; Cao et al., 1999; Liebes et al., 1975; Martenson et al., 1985; Stone et al., 1985). Although there are instances where TMAO has had only minor effects, e.g., the regulatory domain of cystic fibrosis transmembrane conductance regulator (Ostedgaard et al., 2000), its presence seems to be beneficial to most proteins.

The literature on osmolytes inspired us to evaluate the use of TMAO in 2D crystallization experiments with rmMBP, in the hope of reducing the conformational heterogeneity of the protein ensemble and thereby improving the quality of the crystals obtained on lipid monolayers. In addition, we created a mutant of rmMBP in which selected arginyl and lysyl residues were mutated to glutaminyl residues in order to mimic the effects of deimination and reduce the overall net positive charge of the protein, again to facilitate its self-association. In this present paper, we describe the results of 2D crystallization and TEM, circular dichroic (CD) spectroscopic, and dynamic light-scattering (DLS) experiments on the effects of the osmolyte TMAO on the recombinant protein and its mutant. On the basis of these results and of an analysis of amino acid composition, we comment on the potential future efficacy of crystallization strategies for this protein.