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* Universidade de São Paulo-Faculdade de Filosofia, Ciências e Letras de Ribeirão Preto, Ribeirão Preto, Brazil; Instituto de Fisica, Laboratório de Cristalografia Aplicada e Raios X, Universidade Estadual de Campinas, Brazil; Laboratório Nacional de Luz Síncrotron, Campinas, Brazil; Dipartimento di Fisica and Istituto Nazionale per la Fisica della Materia, University of Parma, Parma, Italy; ¶ Dipartimento di Biochimica e Biologia Molecolare, University of Bari, Bari, Italy; and || Dipartimento di Biologia, Difesa e Biotecnologie Agro-forestali, University of Basilicata, Potenza, Italy
Correspondence: Address reprint requests to P. Riccio, E-mail: riccio{at}unibas.it.
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTS AND DISCUSSIONCONCLUDING REMARKSACKNOWLEDGEMENTSREFERENCES |
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTS AND DISCUSSIONCONCLUDING REMARKSACKNOWLEDGEMENTSREFERENCES |
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; Kirschner and Blaurock, 1992; Moscarello, 1996). Myelin basic protein (MBP) is the second major protein in myelin and perhaps the most studied among its components. MBP is important for two reasons: 1), it is believed to have the key role of biological "glue" for the formation, compaction, and maintenance of the multilamellar structure of myelin (Riccio et al., 1986; Readhead et al., 1987); and 2), it is a candidate autoantigen in the context of multiple sclerosis research, since it can induce Experimental Allergic Encephalomyelitis, an animal model of multiple sclerosis (Alvord et al., 1984; Massacesi et al., 1993). Up to the present time, besides the amino-acid sequence and the numerous posttranslational modifications, and a structural model of the 18.5 kDa isoform based in part on electron microscopical data (Beniac et al., 1997; Ridsdale et al., 1997), very little is known about the native three-dimensional protein conformation. One reason is that MBP is usually extracted under rather drastic conditions in a lipid-free, water-soluble form (LF-MBP), and at least partial unfolding must take place (Deibler et al., 1972, 1984). In fact, most studies on the structure of LF-MBP in aqueous solution are in agreement with a flexible coil conformation of the protein (Krigbaum and Hsu, 1975; Gow and Smith, 1989; Smith, 1992). However, the interaction of MBP with detergents, lipids, and other molecules can induce a more ordered structure (Smith, 1982; Haas et al., 1998; Polverini et al., 1999) and lipid-protein interactions are critical for the stability of the myelin sheath (Boggs et al., 1982; Smith, 1992; Staugaitis et al., 1996).
Another way to extract MBP from myelin is to use mild detergents, to maintain the protein in its natural lipidic environment during the purification process (Riccio et al., 1984, 1994). This form is called lipid-bound MBP (LB-MBP). LB-MBP was found to differ in various, mainly immunological, aspects from the lipid-free form (Bobba et al., 1991; Lolli et al., 1993; Massacesi et al., 1993; Liuzzi et al., 1996; Vergelli et al., 1997; Mazzanti et al., 1998). By spectroscopic measurements it was shown that LB-MBP has a much higher proportion of ordered secondary structure than the LF-MBP even after addition of detergents and lipids (Polverini et al., 1999). With LB-MBP and lipids, self-organization of stable, myelinlike membranes could be induced under conditions in which lipids alone remained poorly organized (Riccio et al., 1986, 2000). A comparison between LF-MBP and LB-MBP has been discussed (Riccio and Quagliariello, 1993).
In the present study, to get direct insight into the structures of the lipid-free and the lipid-bound protein forms, we have performed small angle x-ray scattering (SAXS) measurements on LF-MBP and LB-MBP solutions at the National Synchrotron Laboratory in Campinas, Brazil. The experimental data reveal considerable differences between the two protein forms. Moreover, we have modified the current structural model (Beniac et al., 1997; Ridsdale et al., 1997) taking into account circular dichroism (CD) results and theoretical predictions (Polverini et al., 1999). The x-ray scattering data on LB-MBP are in accordance with this new model.
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MATERIALS AND METHODS |
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, 1984). Protein concentration was determined using the Bio-Rad Bradford reagent (Bio-Rad Laboratories, Hercules, CA) and a microassay procedure. MBP was used as a standard, using the molar extinction coefficient 
= 10,300 M-1 at 276.4 nm (Liebes et al., 1975).
Lipid-bound (LB) MBP
LB-MBP was purified as described previously. Briefly, highly purified myelin was treated with the zwitterionic detergent CHAPS (3-((3-cholamido-propyl)dimethylammonio)-1-propane sulfonate) (Boehringer Mannheim, Germany), hydroxyapatite (Bio-Rad Laboratories, Hercules, CA) was used as a filter to separate the nonadsorbed MBP from other myelin proteins, and overnight dialysis was used to remove free lipids from the LB-MBP complex. As the lipid-free counterpart, LB-MBP was electrophoretically pure. Protein purity was also assessed with the MALDI-TOF Pro mass spectrometer of Amersham Biosciences (Freiburg, Germany). Thin-layer chromatography (TLC) showed that the MBP was associated with all native myelin lipids. Relative percentages of lipids in LB-MBP, referred to the values obtained by computerized densitometry of HPTLC plates, were the following (in brackets are shown the percentages of lipids present in whole myelin): cholesterol, 31.5% (44.1%); nonhydroxycerebrosides, 2.5% (5.1%); hydroxycerebrosides, 6.0% (10.9%); sulphatides, 1.5% (7.6%); phosphatidylethanolamine 48.0% (12.8%); phosphatidylserine, 2.5% (3.3%); phosphatidylcholine, 7.0% (3.3%); and sphingomyelin, 1.0% (9.4%). Phosphatidylinositol (3.6% in native myelin) was not detectable in LB-MBP studied in this work.
Structural model of bovine MBP
The three-dimensional homology model of bovine MBP (18.5 kDa isoform) was based on the template of the human 18.5 kDa MBP structure (Ridsdale et al., 1997), using the coordinates available in the Protein Data Bank (Berman et al., 2000), entry ID code 1qcl. The pairwise alignment of human and bovine MBP was performed at the European Bioinformatics Institute using the BLAST network service (Altschul et al., 1997). Where required, single residue substitutions were performed using the tools available in the WHAT IF software (Vriend, 1990). Furthermore, the H10 and G11 residues of human MBP, which are not present in the bovine protein, were cut from the model, and the gap ends were joined with the paste tool in WHAT IF. The bovine Q75 was inserted into the structure and its side-chain conformations were generated using the rotamer library also included in WHAT IF. On the basis of a previous secondary structure prediction and the CD results of Polverini et al. (1999), the two coil segments of bovine MBP corresponding to residues 61–66 and 131–136 were replaced with two small 
-helices, and energy-minimized by means of the Sybyl software package (SYBYL 6.7.1, Tripos, St. Louis, MO). The refined model was evaluated using the WHAT IF tools for protein structure verification.
X-ray scattering experiments
Small angle x-ray scattering experiments were performed at the SAXS beamline of the National Synchrotron Laboratory, Campinas, Brazil (Kellermann et al., 1997). The monochromatic beam was tuned at 7.711 keV. The experimental setup included a temperature-controlled, 1-mm-thick sample cell with thin (30-µm) mica windows and a linear position-sensitive detector. Two sample/detector distances were used, 1600 and 525 mm. Protein samples, containing buffer and detergent, were lyophilized and shipped at ambient temperature from Italy to Brazil. Directly before the x-ray scattering experiments, Milli-Q filtered water (Millipore, Bedford, MA) was added to the lyophilized samples to give the desired protein concentration, and these stock solutions were used directly. Blank (buffer) measurements were performed with solutions containing all the additives used for the protein measurements. The LB-MBP measurements were performed at a protein concentration of 1.28 mg/ml in 20 mM HEPES buffer, pH 7.5, with 0.8% CHAPS. The LF-MBP measurements were performed at a protein concentration of 3.2 mg/ml in 10 mM HEPES buffer, pH 7.5, with 0.4% CHAPS.
All solutions were agitated with a vortexer directly before the measurements. A volume of 
300 µl of solution was necessary for each experiment. The samples were kept at 15°C during the exposures. The time for a single measurement did not exceed 10 min, and the buffer (blank) measurements were performed immediately before or after the protein measurements. In some cases, several cycles of protein and buffer measurements were repeated, and the data were combined for better statistics. Data treatment was performed using the software package TRAT1D (Oliveira et al., 1997). Usual corrections for detector homogeneity, intensity of incident beam, sample absorption, and blank subtraction were included in this routine. The output of this software provides the corrected intensities and error values necessary for data analysis.
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RESULTS AND DISCUSSION |
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sin(
)/
, where is the wavelength used and 2 is the scattering angle). In contrast, the LF-MBP curves did not show such a distinct modulation. The noise was much greater, and the scattering intensity reached zero at lower Q-values than with the LB-MBP.
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). In a plot of the logarithmic scattering intensity ln I(Q) as a function of the square of the momentum transfer, Q, for monodisperse scatterers, the radius of gyration RG is determined using
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0.0005 Å-2. There, from linear regression (solid line), an RG value in the order of 50 Å can be determined. At higher Q2 (0.0015 Å-2), the slope is smaller, corresponding to an RG of 42 Å. These results are, in some respects, similar to those obtained previously by SAXS of LF-MBP under somewhat different environmental conditions (without detergent), where radii of 46 Å (Krigbaum and Hsu, 1975), and of 39 Å (Epand et al., 1974) were determined. The present data indicate clearly that the conformation of LF-MBP is fundamentally different from that of LB-MBP.
Solution structure of LB-MBP
The main objective of the present research was to elucidate the solution structure of LB-MBP. Because the LB-MBP was present in the solution in a monodisperse, compact form, the data permitted further quantitative analysis. As a first step, the pair distribution function of the single protein molecule, p(r) as given by
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) as
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40 Å and a shoulder at 20 Å are visible. As well, the value at larger distances does not directly reach zero until beyond 100 Å. The data are in accordance with a compact protein conformation; however, the protein cannot have a homogeneous globular shape, because then a curve with a single maximum would be expected.
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), the data for LB-MBP were fitted using such a model of concentric spheres, without any other restriction. The fit is shown in Fig. 1 as a dotted line for comparison with the experimental data. In Fig. 4, the curve for the real space model is shown. The electron density profile consists of 10 equidistant steps (shells) of constant electron density. It can be interpreted in terms of an inner sphere with a diameter in the order of 30 Å, where the electron density is higher than that of water, surrounded by a shell of 15 Å thickness, with a density lower than that of water (a profile with inverted signs, such as shown by dotted line in Fig. 4, gives the same fit). Such a conformation could account for the expected organization of the protein (with high electron density), which is surrounded by a shell of lipids and detergent (with low electron density due to the hydrocarbon chains). As well, the dimensions roughly correspond to the expected values. For the protein, the volume can be estimated on the basis of the partial molar volumes of the amino acids (Perkins, 1988) to be 21,000 Å3, corresponding to a compact sphere with a radius of 17 Å. Similarly, the dimension of the outer shell is as expected for a lipid envelope. With the present model, some aspects of the radial density distribution can be derived. However, with this formalism, only strictly centrosymmetric structures can be represented. The differences between the fit (dotted line in Fig. 1) and the experimental data are due to this limitation and they indicate that the protein structure significantly differs from such a centrosymmetric arrangement. To obtain more detailed information on the protein structure, further modeling is necessary.
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. Thus, briefly stated, the protein is represented by an atomic structure composed of N residues. To represent the bounded solvent, the protein is surrounded by a hydration layer of 3 Å thickness, given by a quasiuniform grid of M 
N dummy solvent atoms placed 5 Å outside the protein. The scattering intensity from the chain model, composed of K atoms (K = M + N) averaged by all orientations can be calculated using the Debye formula,
 | (4) |
This approach has the advantage that it is model-free, and a very good agreement with the data can be achieved (solid line in Fig. 1). However, the spatial averaging of the protein orientation causes a dramatic loss of information in a SAXS profile. Also, we have access to just a small region of the reciprocal space and, as a result, a very large number of models can correspond to the same perfect fit. In Fig. 5 (left panel), the result of this model-free fitting is given. The results of five independent fittings, each drawn in a different color, are superimposed. Obviously, all results have some common features. The shapes have some anisotropy and they consist of a curved shape with some branches. The superposition gives the most probable configuration space available for the models as a hint about the possible shape of the protein. It should also be pointed out that in this model, no regions of different electron density can be considered. However, despite these restrictions, these shapes have some striking similarities with results from previous model calculations, where an overall C-shape structure for the human MBP in the presence of lipids was built up using both experimental (electron microscopy) and computational techniques (Beniac et al., 1997; Ridsdale et al., 1997). Starting from this structure (entry 1qcl in Protein Data Bank) and using more recent CD results and refined theoretical predictive methods, a new model for MBP has been obtained. In fact, CD experiments carried out on LB- and LF-MBP (Polverini et al.,1999) clearly showed that the LB form appears to have a consistent amount of ordered secondary structure, while the LF-MBP is a substantially random coil protein. In the same article, theoretical predictions made using different computational methods were shown. These predictions confirmed the tendency of the LB protein to assume ordered secondary structure in accordance with the CD experiments. The main differences with respect to the 1qcl model are in the replacement of two coil segments (residues 61–66 and 131–136), which lie at the two ends of the C-shaped model with -helical structures, although the characteristic C-shape is maintained. In Fig. 5, different orientations of this model (right-hand side) are shown in comparison with the result from the SAXS data analysis (left-hand side).
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CONCLUDING REMARKS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTS AND DISCUSSIONCONCLUDING REMARKSACKNOWLEDGEMENTSREFERENCES |
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), supposing they are structured and stable (Rosenbusch, 2001).
In this article, we have shown the first direct structural information about the conformation of MBP in solution in its native lipidic environment as obtained by small-angle x-ray scattering measurements. A fundamental outcome of this work is that MBP, extracted and purified in its lipidic environment by the procedure as proposed by us, appears suitable for studying the native protein structure and function. The direct isolation of the specific protein-lipid complex is more appropriate than trying to "re-nature" the lipid-free, denatured, protein variety by exposing it to a suitable environment. The data about the conformation of MBP in its native lipidic environment in solution point toward a compact, but not spherical, protein-lipid complex with regions of different electron density. Model-free fitting of the LB-MBP data yielded an extended C-shape for the protein, reminiscent of the predicted structure of the Protein Data Bank. From our further refinement we predict that the protein must be flattened (the C straightens out) in vivo within the major dense line of myelin, where it also interacts with other proteins and where the whole structure is more restrained (cf. Bates and Harauz, 2003). We conclude that even though LB-MBP is still a limited experimental construct, it represents a step in the right direction and an experimentally tractable model system for the natural protein in the myelin membrane.
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ACKNOWLEDGEMENTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTS AND DISCUSSIONCONCLUDING REMARKSACKNOWLEDGEMENTSREFERENCES |
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The major portion of the experimental work was performed at the National Synchrotron Light Laboratory, Brazil, and was sponsored by Coordenação de Aperfeiçoamento de Pessoal de Nível Superior, Brazil, Fundação de Amparo à Pesquisa do Estado de São Paulo (Proc.00/15087-4), Brazil, and Conselho Nacional de Pesquisa, Brazil. Funding from the European Union (Biomed contract BMH4-CT96-0990; Project title: High-Resolution Structures of Myelin Proteins), the Italian Foundation for Multiple Sclerosis, and Istituto Nazionale per la Fisica della Materia is also gratefully acknowledged.
Submitted on May 21, 2003; accepted for publication September 9, 2003.
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REFERENCES |
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