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• W2012079242 abstract "Being intimately involved in cholesterol transport and lipid metabolism human low density lipoprotein (LDL) plays a prominent role in atherogenesis and cardiovascular diseases. The receptor-mediated cellular uptake of LDL is triggered by apolipoprotein B-100 (apoB-100), which represents the single protein moiety of LDL. Due to the size and hydrophobic nature of apoB-100, its structure is not well characterized. Here we present a low resolution structure of solubilized apoB-100. We have used small angle neutron scattering in combination with advanced shape reconstruction algorithms to generate a three-dimensional model of lipid-free apoB-100. Our model clearly reveals that apoB-100 is composed of distinct domains connected by flexible regions. The apoB-100 molecule adopts a curved shape with a central cavity. In comparison to LDL-associated apoB-100, the lipid-free protein is expanded, whereas according to spectroscopic data the secondary structure is widely preserved. Finally, the low resolution model was used as a template to reconstruct a hypothetical domain organization of apoB-100 on LDL, including information derived from a secondary structure prediction. Being intimately involved in cholesterol transport and lipid metabolism human low density lipoprotein (LDL) plays a prominent role in atherogenesis and cardiovascular diseases. The receptor-mediated cellular uptake of LDL is triggered by apolipoprotein B-100 (apoB-100), which represents the single protein moiety of LDL. Due to the size and hydrophobic nature of apoB-100, its structure is not well characterized. Here we present a low resolution structure of solubilized apoB-100. We have used small angle neutron scattering in combination with advanced shape reconstruction algorithms to generate a three-dimensional model of lipid-free apoB-100. Our model clearly reveals that apoB-100 is composed of distinct domains connected by flexible regions. The apoB-100 molecule adopts a curved shape with a central cavity. In comparison to LDL-associated apoB-100, the lipid-free protein is expanded, whereas according to spectroscopic data the secondary structure is widely preserved. Finally, the low resolution model was used as a template to reconstruct a hypothetical domain organization of apoB-100 on LDL, including information derived from a secondary structure prediction. Apolipoprotein B-100 (apoB-100) 2The abbreviations used are: apoB-100, apolipoprotein B-100; LDL, low density lipoprotein; SANS, small angle neutron scattering; VLDL, very low density lipoprotein; IDL, intermediate density lipoprotein; EM, electron microscopy; cryo-EM, cryo-electron microscopy; NSD, normalized spatial discrepancy. is the sole protein component of low density lipoprotein (LDL) and encompasses a variety of functions in lipid metabolism. Like all plasma lipoproteins, LDL takes the shape of a globular particle consisting of a non-polar lipid core surrounded by an amphiphilic coating of protein, phospholipid, and cholesterol (1Muller K. Laggner P. Glatter O. Kostner G. Eur. J. Biochem. 1978; 82: 73-90Crossref PubMed Scopus (52) Google Scholar, 2Laggner P. Kostner G.M. Degovics G. Worcester D.L. Proc. Natl. Acad. Sci. U. S. A. 1984; 81: 4389-4393Crossref PubMed Scopus (28) Google Scholar). LDL is the principal plasma cholesterol carrier and serves as a source of cholesterol for most tissues of the body through receptormediated recognition of apoB-100. From the medical point of view, LDL may become involved in a variety of metabolic disorders, such as hypercholesterolemia, hyperlipidemia, or atherogenesis, which are often linked to a structurally determined dysfunction of apoB-100. Human apoB-100 is a glycoprotein with a molecular mass of about 550 kDa, consisting of 4536 amino acid residues. It is a single chain protein that is associated with hydrophobic molecules in a noncovalent fashion to facilitate their transport and targeting in a hydrophilic environment. The apolipoprotein is synthesized in hepatocytes, where the assembly of the lipoprotein particle takes place, which is mediated by the first 884 amino acid residues of apoB-100 in the presence of a microsomal triglyceride transfer protein (3Shelness G.S. Hou L. Ledford A.S. Parks J.S. Weinberg R.B. J. Biol. Chem. 2003; 278: 44702-44707Abstract Full Text Full Text PDF PubMed Scopus (51) Google Scholar). Furthermore, apoB-100 is a carrier of cell targeting signals that facilitate the uptake of lipoprotein particles by receptor-mediated endocytosis (4Goldstein J.L. Ho Y.K. Basu S.K. Brown M.S. Proc. Natl. Acad. Sci. U. S. A. 1979; 76: 333-337Crossref PubMed Scopus (1952) Google Scholar). A domain close to the COOH-terminal end, which is substantially enriched in basic amino acids (residues 3345-3381), was identified as the receptor binding domain (5Yang C.-Y. Chen S.-H. Gianturco S.H. Bradley W.A. Sparrow J.T. Tanimura M. Li W.-H. Sparrow D.A. DeLoof H. Rosseneu M. Lee F.-S. Gu Z.-W. Gotto Jr., A.M. Chan L. Nature. 1986; 323: 738-742Crossref PubMed Scopus (269) Google Scholar, 6Hospattankar A.V. Law S.W. Lackner K. Brewer Jr., H.B. Biochem. Biophys. Res. Commun. 1986; 139: 1078-1085Crossref PubMed Scopus (30) Google Scholar, 7Knott T.J. Pease R.J. Powell L.M. Wallis S.C. Rall S.C. Innerarity T.L. Blackhart B. Taylor W.H. Marcel Y. Milne R. Johnson D. Fuller M. Lusis A.J. McCarthy B.J. Mahley R.W. Levy-Wilson B. Scott J. Nature. 1986; 323: 734-738Crossref PubMed Scopus (402) Google Scholar). A detailed analysis of the secondary structure by computational methods resulted in the so-called pentapartite model (8Segrest J.P. Jones M.K. Mishra V.K. Anantharamaiah G.M. Garber D.W. Arterioscler. Thromb. 1994; 14: 1674-1685Crossref PubMed Scopus (169) Google Scholar) proposing five consecutive domains with alternating α-helix and β-strand regions. According to this model the predicted domain organization for apoB-100 is NH2-βα1-β1-α2-β2-α3-COOH. The amphipathic nature of these secondary structural elements determines the extent of association with the lipid core. This information was gained by exploring the relative accessibility for trypsin digestion (9Yang C.Y. Kim T.W. Pao Q. Chan L. Knapp R.D. Gotto Jr., A.M. Pownall H.J. J. Protein Chem. 1989; 8: 689-699Crossref PubMed Scopus (19) Google Scholar). The NH2-terminal domain including residues 1-1000 (βα1) was found to be highly trypsin-releasable suggesting that it is not associated with the lipid core in LDL. In addition, this domain contains more than half of the total 25 cysteine residues of apoB-100, all of which are involved in disulfide bonds with other cysteine residues in the same domain. This is a strong indication for a very compact folding of this region, most likely in the form of an independent globular domain that was shown to be highly homologous to the structure of lipovitellin (10Mann C.J. Anderson T.A. Read J. Chester S.A. Harrison G.B. Kochl S. Ritchie P.J. Bradbury P. Hussain F.S. Amey J. Vanloo B. Rosseneu M. Infante R. Hancock J.M. Levitt D.G. Banaszak L.J. Scott J. Shoulders C.C. J. Mol. Biol. 1999; 285: 391-408Crossref PubMed Scopus (170) Google Scholar). The remaining domains (β1-α2-β2-α3) are less characterized and were found to be more closely associated to the lipid core of LDL. Over time several strategies were followed to elucidate the structure and morphology of LDL and apoB-100 on LDL by different biophysical techniques (11Laggner P. Müller K. Q. Rev. Biophys. 1978; 11: 371-425Crossref PubMed Scopus (59) Google Scholar, 12Laggner P. Kostner G.M. Eur. J. Biochem. 1978; 84: 227-232Crossref PubMed Scopus (34) Google Scholar, 13Walsh M.T. Atkinson D. J. Lipid Res. 1990; 31: 1051-1062Abstract Full Text PDF PubMed Google Scholar, 14Lund-Katz S. Laplaud P.M. Phillips M.C. Chapman M.J. Biochemistry. 1998; 37: 12867-12874Crossref PubMed Scopus (117) Google Scholar). The mapping of apoB-100 on the surface of LDL by triangulation techniques with monoclonal antibodies generated the generally accepted idea that the protein is a belt wrapped once around the LDL particle (15Chatterton J.E. Phillips M.L. Curtiss L.K. Milne R.W. Marcel Y.L. Schumaker V.N. J. Biol. Chem. 1991; 266: 5955-5962Abstract Full Text PDF PubMed Google Scholar, 16Chatterton J.E. Phillips M.L. Curtiss L.K. Milne R. Fruchart J.C. Schumaker V.N. J. Lipid Res. 1995; 36: 2027-2037Abstract Full Text PDF PubMed Google Scholar). Studies using cryoelectron microscopy (cryo-EM) in vitreous ice resulted in a three-dimensional low resolution model of the LDL particle (17Orlova E.V. Sherman M.B. Chiu W. Mowri H. Smith L.C. Gotto A.M. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 8420-8425Crossref PubMed Scopus (117) Google Scholar). Nevertheless, despite the successful crystallization of LDL (18Prassl R. Chapman J.M. Nigon F. Sara M. Eschenburg S. Betzel C. Saxena A. Laggner P. J. Biol. Chem. 1996; 271: 28731-28733Abstract Full Text Full Text PDF PubMed Scopus (15) Google Scholar, 19Ritter S. Frey I. Diederichs K. Grathwohl D. Keul J. Baumstark M.W. Proteins Struct. Funct. Genet. 1997; 28: 293-297Crossref PubMed Scopus (16) Google Scholar, 20Lunin V.Y. Lunina N.L. Ritter S. Frey I. Berg A. Diederichs K. Podjarny A.D. Urzhumtsev A. Baumstark M.W. Acta Crystallogr. D. Biol. Crystallogr. 2001; 57: 108-121Crossref PubMed Scopus (32) Google Scholar), a high resolution structure of apoB-100 in LDL is not available yet and awaits further clarification. Other approaches were applied to investigate the morphology of apoB-100 after extraction from its native lipid environment. Several detergents were used to solubilize apoB-100, and the resulting protein-detergent complex was subjected to negative staining EM and cryo-EM in vitreous ice (21Zampighi G. Reynolds J.A. Watt R.M. J. Cell Biol. 1980; 87: 555-561Crossref PubMed Scopus (15) Google Scholar, 22Ikai A. J. Biochem. (Tokyo). 1980; 88: 1349-1357Crossref PubMed Scopus (13) Google Scholar, 23Gantz D.L. Walsh M.T. Small D.M. J. Lipid Res. 2000; 41: 1464-1472Abstract Full Text Full Text PDF PubMed Google Scholar, 24Walsh M.T. Atkinson D. Biochemistry. 1983; 22: 3170-3178Crossref PubMed Scopus (41) Google Scholar, 25Phillips M.L. Schumaker V.N. J. Lipid Res. 1989; 30: 415-422Abstract Full Text PDF PubMed Google Scholar). To a certain extent, these results differ depending on preparation procedure and imaging technique used. Curved shapes and horseshoe or thread-like morphologies were found. Flexible strings up to 1000 Å long, with width varying from 20 to 70 Å, were imaged. However, a common feature in all studies is that lipid-free apoB-100 adopts an elongated conformation. As outlined above, the most conclusive experimental results on the structure of apoB-100 are reported from EM imaging, but they lack three-dimensional information. To address this issue, we have applied small angle neutron scattering (SANS) combined with the contrast matching technique to elucidate three-dimensional characteristics of lipid-free apoB-100 and to assess its structural flexibility at the same time. We have restored a low resolution model of apoB-100 while eliminating contributions of the solubilizing detergent. Finally, based on our low resolution structure model in combination with a secondary structure prediction, a hypothetical arrangement of distinct modules of apoB-100 within LDL is proposed. Preparation of LDL from Human Plasma—Blood was taken from normolipidemic, single donors. To prevent clotting, oxidation, microbial degradation, and proteolytic cleavage, 1 mg/ml Na2EDTA, 0.05% gentamycin sulfate (Serva, Heidelberg, Germany) and a protease inhibitor mixture (Roche Diagnostics, Basel, Switzerland) were added. Whole blood was centrifuged at 4500 rpm, 10 min, 5 °C, and the supernatant was carefully separated from the pellet and stored in aliquots at -70 °C after addition of 10% (w/v) sucrose as a cryoprotectant. LDL (density range 1.02-1.063) was prepared by ultracentrifugation and dialyzed at 4 °C against standard buffer (10 mm sodium phosphate, 1 mg/ml Na2EDTA, 0.05% gentamycin sulfate, pH 7.4). LDL samples were assessed for protein concentration by the BCA method (BCA protein assay kit, Pierce) Purity and homogeneity were determined by SDS-PAGE. Solubilization and Purification of ApoB-100—Delipidation of LDL was performed by modified procedures previously described by Watt and Reynolds (26Watt R.M. Reynolds J.A. Biochemistry. 1980; 19: 1593-1598Crossref PubMed Scopus (15) Google Scholar) and Melnik and Melnik (27Melnik B.C. Melnik S.F. Anal. Biochem. 1988; 171: 320-329Crossref PubMed Scopus (5) Google Scholar). Briefly, the non-ionic detergent Nonidet P-40 (Roche Applied Science, Penzberg, Germany) at 55 mg/mg of LDL protein was added to LDL typically containing 8-10 mg of LDL protein in a total volume of 3 ml and stored under argon. After a minimum of 2 h of mild stirring in the dark at 4 °C, the solution was applied to a 1.5 × 50-cm column of Sepharose CL-6B (Sigma) equilibrated with an eluent buffer (0.5 mm Nonidet P-40 in standard buffer). Fractions were collected at a flow rate of 0.66 ml/min while measuring the absorbance at 280 nm. All fractions from the first peak after the void volume (clear colorless solution) were pooled and brought to protein concentrations between 4.0 and 7.0 mg/ml using Amicon Ultra-15 centrifugal filter units (Millipore, Billerica, MA) with 100 kDa molecular mass cutoff. Excess detergent was slowly removed from concentrated samples by dialysis for 24 h against standard buffer containing a suspension of Amberlite XAD-2 hydrophobic porous beads (Sigma) and using a SpectraPor CE dialysis membrane with 300-kDa molecular mass cutoff (Spectrum Laboratories, Inc., Breda, The Netherlands). To make sure that delipidation was complete, the concentrations of total cholesterol and phospholipid were determined by use of enzymatic assay kits “Cholesterol C” and “Phospholipids B” (Wako, Neuss, Germany), respectively. The amounts of cholesterol and phospholipid found in the delipidated sample were negligible. The protein concentration was determined by the BCA method, and purity and homogeneity of solubilized apoB-100 were assessed by SDS-PAGE. The concentration of Nonidet P-40 was determined by absorbance measurements at 280 nm. The absorption coefficient for Nonidet P-40 was calculated from linear regression of a standard dilution series in deionized water. The theoretical absorption from the protein was calculated from the amino acid composition (28Pace C.N. Vajdos F. Fee L. Grimsley G. Gray T. Protein Sci. 1995; 4: 2411-2423Crossref PubMed Scopus (3472) Google Scholar) and subtracted from absorption values of samples containing both protein and Nonidet P-40 to give accurate values for detergent concentration. Circular Dichroism Spectroscopy—Far-UV/CD spectra were obtained in a wavelength range from 190 to 250 nm with a Jasco J-715 spectropolarimeter (Jasco Inc., Easton, MD) equipped with a personal computer. Each spectrum was recorded as an average of three scans taken with the following parameters: step resolution, 0.5 nm; scan speed, 50 nm/min; response, 1 s; and bandwidth, 1 nm. The spectra were base line-corrected by subtracting the signal of detergent in standard buffer. The results were expressed in terms of mean residue ellipticity [θ] (degree·cm2·dmol-1) defined as follows. [θ]=θobs10·c·l(Eq. 1) θobs is the measured ellipticity in degrees, c is the protein residue concentration in mol/liter, and l is the length of the light path in cm. The ellipticity data were analyzed for secondary structure by a non constrained least squares analysis using the CDSSTR algorithm based on reconstruction of experimental data (29Lobley A. Whitmore L. Wallace B.A. Bioinformatics. 2002; 18: 211-212Crossref PubMed Scopus (645) Google Scholar, 30Whitmore L. Wallace B.A. Nucleic Acids Res. 2004; 32: W668-W673Crossref PubMed Scopus (2001) Google Scholar). SANS Experiments—Neutron scattering data were obtained at beam lines D11 and D22 using the high flux reactor at the Institut Laue-Langevin (Grenoble, France). To determine the contrast match point of the detergent contrast variation experiments were performed on instrument D11. Scattering profiles were recorded of 28 mm Nonidet P-40 dissolved in buffer at several D2O/H2O ratios (0, 10, 20, 30, 40, and 100% D2O). This detergent concentration, which is about 10 times the critical micellar concentration, was chosen to be the same as in the apoB-100-Nonidet P-40 complex used for further SANS experiments. Sample-detector distances of 1.2, 3, and 12 m (collimations 5.5, 5.5, and 13.5 m), circular cells with a thickness of 2 mm, and a neutron wavelength of 6 Å(Δλ/λ of 10%) were used. The contrast match point is given by the D2O concentration at which the intensity of forward scattering approaches zero, I(0) = 0. I(0) values were determined by extrapolation of the scattering profiles to zero angle using the Guinier approximation (31Guinier A. Fournet G. Small Angle Scattering of X-rays. John Wiley & Sons, New York1955Google Scholar). The normalized intensities (I(0)/c)1/2 were plotted as a function of D2O content (data not shown). The intercept of this linear dependence on the abscissa indicates the contrast match point of the detergent. In case of Nonidet P-40, the scattering intensity was matched at 18% D2O. The apoB-100-Nonidet P-40 complex was measured at the match point concentration of 18% D2O/H2O on instrument D22 at a protein concentration of 6.4 mg/ml and a detergent concentration of 28 mm using a rectangular cell with a thickness of 2 mm positioned in a thermostatted sample rack at 8 °C. The typical acquisition time was 30 min. The exposure of the sample was repeated 12 times to verify sample stability. Sample-detector/collimation distances of 14.0/14.4 m and 2.8/2.8 m, a wavelength λ of 6 Å(Δλ/λ of 10%), and a rectangular beam aperture of 16 × 16 mm were used. To correct for background scattering the Nonidet P-40/buffer curve measured at 18% D2O/H2O was subtracted. Data reduction was based on standard ILL software using the programs DETEC, RNILS, SPOLLY, RGUIM, and RPLOT (32Ghosh R.E. A Computing Guide for Small Angle Scattering Experiments. Institut Laue-Langevin, Grenoble, France1989Google Scholar). The data acquired at both sample-detector distances were merged resulting in a total q-range of 0.004-0.32 Å-1 (q = 4πsinθ/λ, 2θ is the scattering angle) used for further calculations. Analysis of Reduced Scattering Data—Radius of gyration (RG) of apoB-100 was determined by the Guinier approximation from the low q-regions (q·RG ≤ 1.3) of the scattering profiles (31Guinier A. Fournet G. Small Angle Scattering of X-rays. John Wiley & Sons, New York1955Google Scholar) according to the following equation. ln(l(q))≅ln(l(0))-RG2·q23(Eq. 2) At small angles when plotted as log(I(q)) versus q2 the scattering profile should be linear although this relationship is rigorously valid only for q ≤ RG-1(33Feigin L.A. Svergun D.I. Structural Analysis by Small-angle X-ray and Neutron Scattering. Plenum Press, New York1987Crossref Google Scholar) The pair-distance distribution function p(r), p(r)=12·π2∫0∞r·q·l(q)·sin(r·q)·dq(Eq. 3) was obtained by indirect Fourier transformation of the scattered intensities I(q) using the program GNOM (34Svergun D.I. J. Appl. Crystallogr. 1992; 25: 495-503Crossref Scopus (2987) Google Scholar). The p(r) corresponds to the distribution of distances r between any two volume elements within one particle weighted by the product of their scattering length density relative to that of the solvent. This offers an alternative calculation of RG that is based on the full scattering curve and gives the maximum dimension of the macromolecule Dmax, as the distance where the p(r) function approaches zero (35Glatter O. J. Appl. Crystallogr. 1977; 10: 415-421Crossref Google Scholar). Ab Initio Modeling—The low resolution shape of apoB-100 was restored from experimental data using the program DAM-MIN (36Svergun D.I. Biophys. J. 1999; 76: 2879-2886Abstract Full Text Full Text PDF PubMed Scopus (1756) Google Scholar). The scattering curve up to qmax = 0.25 Å-1 was used for fitting corresponding to a resolution of ∼25Å (2π/qmax). DAMMIN calculates the scattering intensities from a multiphase model of a particle constructed of a finite number of dummy beads. The beads are characterized by a configuration vector (X) assigning them to a phase or to the solvent. The program starts searching for a model in a volume filled by hexagonally packed uniform beads. The dimensions were selected according to the resolution of the scattering profile while the overall dimension of the initial search object is taking into account. DAMMIN searches for the best model configuration minimizing the discrepancy function (f(X) = χ2 + α·P(X)) using the simulated annealing method (37Kirkpatrick S. Gelatt Jr., C.D. Vecci M.P. Science. 1983; 220: 671-680Crossref PubMed Scopus (31663) Google Scholar), where χ is the discrepancy between the calculated and experimental curve, and α·P(X) is a looseness penalty with positive weight α > 0. The aim of this method is to randomly modify the coordinates of the beads while always approaching the configurations that decrease the energy f(X). 3For further details the reader is referred to the web site www.embl-hamburg.de/ExternalInfo/Research/Sax/dammin.html showing an animation of this fitting approach. The bead radius used for the ab initio modeling of solubilized apoB-100 was set to 14 Å. This value was chosen due to experimental results (Dmax = 600 Å, resolution ∼25 Å) and certain constraints within the program DAMMIN. Different independent models with similar goodness of fit were obtained. Ten of these models were used for automated averaging by the program DAMAVER (38Volkov V.V. Svergun D.I. J. Appl. Crystallogr. 2003; 36: 860-864Crossref Scopus (1620) Google Scholar). This program computes the values for the normalized spatial discrepancy (NSD) between each pair in the set. The average NSD value for each model was calculated with respect to the rest of the set and the model with the lowest NSD was selected as reference model. All other models were superimposed onto the reference model using the program SUPCOMB (39Kozin M.B. Svergun D.I. J. Appl. Crystallogr. 2001; 34: 33-41Crossref Scopus (1093) Google Scholar). Finally, the entire assembly of beads was remapped onto a densely packed grid of beads where each grid point was characterized by its occupancy factor. The portion of points with higher non-zero occupancy was selected to yield the volume equal to the average excluded volume of the models. Secondary Structure Prediction—The complete amino acid sequence of apoB-100 was subjected to the automated secondary structure prediction service SSpro 2.0 using bidirectional recurrent neural networks (40Pollastri G. Przybylski D. Rost B. Baldi P. Proteins Struct. Funct. Genet. 2002; 47: 228-235Crossref PubMed Scopus (623) Google Scholar). The results of this prediction were visualized using POLYVIEW (41Porollo A.A. Adamczak R. Meller J. Bioinformatics. 2004; 20: 2460-2462Crossref PubMed Scopus (121) Google Scholar). Circular Dichroism Spectroscopy—To assess the effects of delipidation on protein folding, circular dichroism spectra of both solubilized apoB-100 and native LDL were recorded and evaluated. Secondary structure contents were calculated from the normalized spectra (Fig. 1) and expressed as percent secondary structure as shown in Table 1.TABLE 1Secondary structure calculated from circular dichroic spectra by the CDSSTR methodSampleα-Helixβ-StrandTurnsCoilNRMSDaThe NRMSD value indicates the root mean square deviation of fits to measured spectra.LDL37%17%20%25%0.010ApoB-10040%21%19%16%0.007PredictedbResults are from the secondary structure prediction. This algorithm is not capable of discerning turns and coil structures, thus a sum of both is given.36%23%41%a The NRMSD value indicates the root mean square deviation of fits to measured spectra.b Results are from the secondary structure prediction. This algorithm is not capable of discerning turns and coil structures, thus a sum of both is given. Open table in a new tab These results are in agreement with a secondary structure prediction, and the values obtained for native LDL are consistent with data reported previously (42Walsh M.T. Atkinson D. J. Lipid Res. 1986; 27: 316-325Abstract Full Text PDF PubMed Google Scholar). The content of α-helix and β-strands of detergent-solubilized apoB-100 appears slightly higher than that of native LDL, while the contents of turns and coil structures seem to be slightly lower. However, within the experimental error margin, the overall secondary structure of Nonidet P-40 solubilized apoB-100 is well conserved. Similar results were reported by Watt and Reynolds (26Watt R.M. Reynolds J.A. Biochemistry. 1980; 19: 1593-1598Crossref PubMed Scopus (15) Google Scholar) for apoB-100 solubilized by n-dodecyl octaethylene glycol monoether, a detergent that is closely related to Nonidet P-40, although due to the choice of a different evaluation method, the absolute values in the results vary. SANS Data—The final scattering curve was obtained by merging the scattering profiles of apoB-100 solubilized in Nonidet P-40 collected at two sample-detector distances (Fig. 2A). The RG value derived from the Guinier approximation was 150 ± 4 Å (Fig. 2A, inset). The linearity of the fit suggests that the data accurately reflect the non-aggregated state of the protein in solution. To verify this, the molecular weight of the protein was calculated. In a two-component system, the molecular weight of a particle in solution is proportional to I(0) according to the following equations (43Jacrot B. Zaccai G. Biopolymers. 1981; 20: 2413-2426Crossref Scopus (290) Google Scholar, 44Zaccai G. Jacrot B. Annu. Rev. Biophys. Bioeng. 1983; 12: 139-157Crossref PubMed Scopus (114) Google Scholar), l(0)=c·NAM∑i(bi-ρ0·vi)2(Eq. 4) whereas ∑i(bi-ρ0·vi)=MNA(B-ρ0·v¯).(Eq. 5) M is the molecular mass, c stands for the protein concentration, bi is the scattering length contained in a small volume, ν, ρ0 is the scattering length density of the solvent, B is the scattering length per unit mass, and v̄ represents the partial specific volume of the macromolecule. The amount of exchangeable protons was assumed to be 80% resulting in a B value of 2.55·10-14 cm. The scattered intensity at zero angle I(0) was obtained by extrapolation of the Guinier plot to q2 → 0. I(0) was found to be 0.61 cm-1. The molecular mass calculated from the zero angle intensity was 547,000 Da, somewhat higher than 512,816 Da as calculated from the amino acid sequence. The discrepancy between these values can be explained by the contribution of glycosylated residues accounting for up to 7% of the molecular mass and a certain error in the protein concentration. The protein concentration is generally considered critical for the calculation of molecular weight by scattering methods. For our samples c was determined by a colorimetric assay using bovine serum albumin as a reference standard, as a direct measurement was not feasible due to interference from the detergent near the absorption maximum of 280 nm. Assuming an elongated particle shape, the mean radius of gyration of the cross-section Rxs and the mean cross-sectional intensity at zero angle [I(q)q]q→0 are obtained from the following equation. lnlq·q≅lnlq·q→0-Rxs2·q22(Eq. 6) Applying this formalism to our results, two linear regions were identified in separate q ranges, q = 0.0075-0.016 Å-1 and q2 = 0.017-0.04 Å-1 (Fig. 2B). The cross-sectional radii of gyration Rxs1 = 80 ± 17 Å and Rxs2 = 33 ± 8 Å were obtained from the slopes of the linear regressions. In general, elongated and multimodular proteins with intrinsic flexibility exhibit a cross-sectional plot with two regions, a steeper innermost one and a flatter outermost one (45Pilz I. Kratky O. Licht A. Sela M. Biochemistry. 1973; 12: 4998-5005Crossref PubMed Scopus (61) Google Scholar). This applies particularly for factor H (46Aslam M. Perkins S.J. J. Mol. Biol. 2001; 309: 1117-1138Crossref PubMed Scopus (118) Google Scholar), immunoglobulins IgA1 (47Boehm M.K. Woof J.M. Kerr M.A. Perkins S.J. J. Mol. Biol. 1999; 286: 1421-1447Crossref PubMed Scopus (198) Google Scholar) and IgA2 (48Furtado P.B. Whitty P.W. Robertson A. Eaton J.T. Almogren A. Kerr M.A. Woof J.M. Perkins S.J. J. Mol. Biol. 2004; 338: 921-941Crossref PubMed Scopus (92) Google Scholar). For apo-B100 we can assume that the innermost steeper part of the cross-sectional plot represents the isotropic cross-section of the overall dimension whereas the outer flatter part reflects the anisotropic/modular organization of the protein (49Pilz I. Glatter O. Kratky O. Small Angle X-ray Scattering. Academic Press, New York1982: 239-293Google Scholar). The values RG, Rxs1, and Rxs2 were used to analyze the axial dimension of apoB-100. The length of an elongated elliptical cylinder can be calculated according to Glatter (50Glatter O. Glatter O. Kratky O. Small Angle X-ray Scattering. Academic Press, New York1982: 119-196Google Scholar), L=12·(RG2-Rxs2)(Eq. 7) yielding an estimated particle length of L = 440-507 Å for apo-B100. For an ideal elliptical cylinder this particle length L should be reflected in the maximum dimension Dmax from the p(r) function (Fig. 2C). The p(r) shows a tailing with a Dmax of 600 Å typical for an elongated particle, however, significantly longer as calculated from the Guinier radii of gyration. Accordingly, the calculation of RG from the p(r) function gives a value of 165 ± 10 Å, also higher as the value obtained from the Guinier approximation RG = 150 ± 4 Å. However, taking into consideration difficulties associated with the determination of such large RG values, they can be considered to be in good agreeme" @default.
• W2012079242 created "2016-06-24" @default.
• W2012079242 creator A5007867266 @default.
• W2012079242 creator A5029196723 @default.
• W2012079242 creator A5032701796 @default.
• W2012079242 creator A5060111068 @default.
• W2012079242 creator A5067755174 @default.
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• W2012079242 date "2006-07-01" @default.
• W2012079242 modified "2023-10-13" @default.
• W2012079242 title "Modular Structure of Solubilized Human Apolipoprotein B-100" @default.
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