Effects of the osmolyte trimethylamine-N-oxide on conformation, self-association, and two-dimensional crystallization of myelin basic protein
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.
Section snippets
Materials and methods
Materials. Electrophoresis-grade acrylamide, ultrapure TRIS-free base, and ultrapure EDTA were purchased from ICN Biomedicals (Costa Mesa, CA). Electrophoresis-grade sodium dodecyl sulphate (SDS) was purchased from Bio-Rad Laboratories (Mississauga, Ontario, Canada). The Ni2+-NTA agarose beads were obtained from Qiagen (Mississauga, Ontario, Canada). Unless otherwise specified, most other chemicals were reagent grade and purchased from either Fisher Scientific (Unionville, Ontario, Canada) or
Two-dimensional crystallization experiments
The crystallization experiments in this study were founded upon earlier work (Ishiyama, 2000; Ishiyama et al., 2000, Ishiyama et al., 2001) that produced planar, close-packed hexagonal arrays using rmMBP deiminated to an average of 9 deiminations/molecule on a 1:1 Ni2+-NTA-DOGS:PI monolayer. Using this system as a starting point, crystallization trials of rmMBP were performed with monolayers containing PI (Fig. 1a) and PI(4)P (Fig. 1b). Planar, closely packed arrays of protein were formed with
Acknowledgements
The authors are grateful to Dr. Wayne Bolen for advice on the preparation and use of TMAO, Dr. Dawn Larson for comments on the manuscript, and Drs. Mario Moscarello and Denise Wood for their support. This work was supported by the Natural Sciences and Engineering Research Council of Canada (GH, FRH) and by the Multiple Sclerosis Society of Canada (GH).
References (91)
- et al.
Forcing thermodynamically unfolded proteins to fold
J. Biol. Chem.
(1998) - et al.
Trimethylamine N-oxide-induced cooperative folding of an intrinsically unfolded transcription-activating fragment of human glucocorticoid receptor
J. Biol. Chem.
(1999) - et al.
Characterization of a recombinant murine 18.5 kDa myelin basic protein
Prot. Expression Purif.
(2000) - et al.
An Arg/Lys → Gln mutant of recombinant murine myelin basic protein as a mimic of the deiminated form implicated in multiple sclerosis
Prot. Expression Purif.
(2002) - et al.
Three-dimensional structure of myelin basic protein. I. Reconstruction via angular reconstitution of randomly oriented single particles
J. Biol. Chem.
(1997) - et al.
Cryoelectron microscopy of protein–lipid complexes of human myelin basic protein charge isomers differing in degree of citrullination
J. Struct. Biol.
(2000) - et al.
The osmophobic effect: Natural selection of a thermodynamic force in protein folding
J. Mol. Biol.
(2001) - et al.
Coupling of folding and binding for unstructured proteins
Curr. Opin. Struct. Biol.
(2002) - et al.
Structure analysis of soluble proteins using electron crystallography
Micron
(2001) - et al.
Experimental allergic encephalomyelitis. An encephalitogenic basic protein from bovine brain
Arch. Biochem. Biophys.
(1969)
No effect of trimethylamine-N-oxide on the internal dynamics of the protein native fold
Biophys. Chem.
2D crystallization of membrane proteins: rationales and examples
J. Struct. Biol.
The effects of deimination of myelin basic protein on structures formed by its interaction with phosphoinositide-containing lipid monolayers
J. Struct. Biol.
The formation of helical tubular vesicles by binary monolayers containing a nickel-chelating lipid and phosphoinositides in the presence of basic polypeptides
Chem. Phys. Lipids
On the crystallization of proteins
J. Mol. Biol.
Two-dimensional crystallization of histidine-tagged, HIV-1 reverse transcriptase promoted by a novel nickel-chelating lipid
J. Struct. Biol.
The conformation of the glucocorticoid receptor af1/tau1 domain induced by osmolyte binds co-regulatory proteins
J. Biol. Chem.
The stabilization of proteins by sucrose
J. Biol. Chem.
Two-dimensional crystallization on lipid layer: a successful approach for membrane proteins
J. Struct. Biol.
Two-dimensional crystallization of membrane proteins: the lipid layer strategy
FEBS Lett.
Structural basis for the binding of an immunodominant peptide from myelin basic protein in different registers by two HLA-DR2 proteins
J. Mol. Biol.
Crystal structure of a superantigen bound to the high-affinity, zinc-dependent site on MHC class II
Immunity
Solution behavior, circular dichroism, 220 MHz PMR studies of the bovine myelin basic protein
Biochim. Biophys. Acta
Myelin basic protein: what does it do?
Two-dimensional crystallogenesis of transmembrane proteins
Micron
Conditions of two-dimensional crystallization of cholera toxin B-subunit on lipid films containing ganglioside GM1
J. Struct. Biol.
31P and 1H NMR studies of the effect of the counteracting osmolyte trimethylamine-N-oxide on interactions of urea with ribonuclease A
J. Biol. Chem.
Osmolytes stabilize ribonuclease S by stabilizing its fragments S protein and S peptide to compact folding-competent states
J. Biol. Chem.
Three-dimensional structure of myelin basic protein. II. Molecular modelling and considerations of predicted structures in multiple sclerosis
J. Biol. Chem.
The natural osmolyte trimethylamine-N-oxide (TMAO) restores the ability of mutant tau to promote microtubule assembly
FEBS Lett.
Trimethylamine-N-oxide-induced folding of α-synuclein
FEBS Lett.
The next generation of the IMAGIC image processing system
J. Struct. Biol.
Electron crystallography of two-dimensional crystals of membrane proteins
J. Struct. Biol.
Intrinsically unstructured proteins: re-assessing the protein structure–function paradigm
J. Mol. Biol.
Manipulating the amyloid-beta aggregation pathway with chemical chaperones
J. Biol. Chem.
Isolation of myelin basic protein from equine spinal cord under non-denaturing conditions
J. Neurochem.
Preferential interactions determine protein solubility in three-component solutions: the magnesium chloride system
Biochemistry
Different tools to study interaction potentials in γ-crystallin solutions: relevance to crystal growth
Acta Crystallogr
The pathobiology of myelin mutants reveals novel biological functions of the MBP and PLP genes
Brain Pathol.
Rapid release and unusual stability of immunodominant peptide 45–89 from citrullinated myelin basic protein
Biochemistry
Structural and functional implications of tau hyperphosphorylation: Information from phosphorylation-mimicking mutated tau proteins
Biochemistry
New insights on the biology of myelin basic protein gene: the neural–immune connection
J. Neurosci. Res.
Probing the conformation of a human apolipoprotein C-1 by amino acid substitutions and trimethylamine-N-oxide
Prot. Sci.
Particle size analysis: number distributions by dynamic light scattering
Can. J. Spectrosc.
Analogous structural motifs in myelin basic protein and in MARCKS
Mol. Cell. Biochem.
Cited by (49)
An intrinsically disordered protein in F127 hydrogel: Fluorescence correlation spectroscopy and structural diversity of beta casein
2021, Chemical Physics LettersCitation Excerpt :In another study, Chattopadhyay and group applied FCS to understand the aggregation pathway of an IDP, α-synuclein [20]. Osmolyte induced self-association of myelin, an IDP [21] and solvent induced collapse of α-synuclein were recently reported [22]. Mukhopadhyay, Datta and co-workers applied ultrafast solvation to study slow water confined in an IDP κ-casein [23,24].
Myelin basic protein dynamics from out-of-equilibrium functional state to degraded state in myelin
2020, Biochimica et Biophysica Acta - BiomembranesThe Proline/Glycine-Rich Region of the Biofilm Adhesion Protein Aap Forms an Extended Stalk that Resists Compaction
2017, Journal of Molecular BiologySubstitutions mimicking deimination and phosphorylation of 18.5-kDa myelin basic protein exert local structural effects that subtly influence its global folding
2016, Biochimica et Biophysica Acta - BiomembranesCitation Excerpt :Here, however, the density of lateral compaction of MBP on a membrane surface does not appear to be altered by pseudo-deimination as ascertained by DEER spectroscopy [36]. Nevertheless, it has been demonstrated previously that this modification alters both the geometry of protein–protein ordering and the degree of membrane adhesion significantly, and the interactions of MBP with cytoskeletal proteins and calcium-activated calmodulin (e.g., [10,18,19,21,25,27,33,53,54,69–72]). In earlier electron microscopy studies of two-dimensional arrays of MBP variants formed on lipid monolayers, pseudo-deimination at 6 sites and enzymatic deimination at an average of 9 sites resulted in different kinds of ordering, a switch from closely-packed fibrils to what appeared to be hexagonal close-packing [69,70,73].
Regulatory effect of the glial Golli-BG21 protein on the full-length murine small C-terminal domain phosphatase (SCP1, or Golli-interacting protein)
2014, Biochemical and Biophysical Research CommunicationsLateral self-assembly of 18.5-kDa myelin basic protein (MBP) charge component-C1 on membranes
2012, Biochimica et Biophysica Acta - BiomembranesCitation Excerpt :Less is known about inter-protein interactions within myelin, although it has been investigated for some time [28–30]. Cryo-transmission electron microscopic (TEM) studies of two-dimensional arrays of hexahistidine-tagged MBP on artificial lipid monolayers revealed structures with a diameter of about 3 nm–4 nm, and lamellar-like arrangements featuring characteristic 4.8 nm repeats [2,31–33]. This method features a characteristic resolution of several nanometers, or requires the existence of intermediate to long-range order.