Statistical conformation of human plasma fibronectin

Fibronectin is a multifunctional glycoprotein (molecular mass, M = 530 kg/mol) of the extra cellular matrix (ECM) having a major role in cell adhesion. In physiological conditions, the conformation of this protein still remains debated and controversial. Here, we present a set of results obtained by scattering experiments. In "native" conditions, the radius of gyration (R(g) = 15.3 +/- 0.3 nm) was determined by static light scattering as well as small-angle neutron scattering. The hydrodynamic radius (R(H) = 11.5 +/- 0.1 nm) was deduced from quasi-elastic light scattering measurements. These results imply a low internal concentration compared to that of usual globular proteins. This is also confirmed by the ratio R(H)/R(g) = 0. 75 +/- 0.02 consistent with a Gaussian chain, whereas R(H)/R(g) = 1. 3 for spherical shaped molecules. However, adding a denaturing agent (urea 8 M) increases R(g) by a factor 2. This means that fibronectin "native" chain is not either completely unfolded. The average shape of fibronectin conformation was also probed by small-angle neutron scattering performed for reverse scattering vector q(-)(1) smaller than R(g) (0.2 < q(-)(1) < 15 nm). The measured form factor is in complete agreement with the form factor of a random string of 56 beads of 5 nm diameter. It rules out the possibility of unfolded chain as well as globular structures. These results have structural and biological implications as far as ECM organization is concerned.     

Protein S1 from Escherichia coli ribosomes: an improved isolation procedure and shape determination by small-angle X-ray scattering.

Ribosomal protein S1 from Escherichia coli was studied in solution by small-angle X-ray scattering and the following parameters were obtained. The radius of gyration R = 8.0 +/- 0.2 nm; largest diameter D = 28 nm; molecular weight = (8--9) x 10(4). The data also yielded (with the assumption of a rigid particle with almost constant electron density) two radii of gyration of cross-section Rq1 = 2.5 +/- 0.1 nm and Rq2 = 1.05 +/- 0.05 nm and molecular volume = 140 nm3. The experimental scattering curve of S1 was compared with the theoretical scattering curves for several rigid triaxial homogeneous bodies and the closest fit was given by that of a flat elliptical cylinder with the dimensions of 4.5 nm and 0.88 nm for the two semiaxes and 26.5 nm for height. The results from the present X-ray scattering studies and those from limited proteolytic digestion of protein S1 [J. Mol. Biol. 127, 41--54, (1979)] support the notion that the structure of protein S1 is organized into two distinct subdomains within its elongated overall shape. Protein S1 was purified for this study by an efficient procedure which yielded 12 mg S1/g ribosomes. The isolated protein was fully active in functional tests both before and after X-ray irradiation.     

Small-angle X-ray studies of the human immunoglobulin molecule Kol.

The conformation of the human immunoglobulin molecul Kol [IgG I, kappa2 gamma2, Gm(f)+] was studied by small-angle X-ray scattering in 0.15 M NaCl solution. The radius of gyration was found to be 5.84 +/- 0.04 nm, the volume 329 +/- 15 nm3 and the molecular weight 150 000 +/- 10 000. Information on the overall shape was obtained by comparing the experimental scattering curve with the calculated curves for various models. The models were obtained by arranging the models found for the Fab and Fc fragments of the same immunoglobulin molecule in a different manner. A model which fits all the date and the form of the experimental scattering curve is presented.     

Conformation of the ribosomal protein S1 of Thermus thermophilus in solution under different ionic conditions

The structure of protein SI of Thermus thermophilus (M = 61 kDa) in solution at low and moderate ionic strengths (0 M and 100 mM NaCl, respectively) has been studied by small-angle X-ray and neutron scattering. It was found that protein S1 has a globular conformation under both ionic conditions. The modelling of different packing of six homologous domains of S1 on the basis of the NMR-resolved structure of one domain showed that the best fit of calculated scattering patterns from such complexes to experimental ones is observed at a compact package of the domains. The calculated value of the radius of gyration of the models is 28-29 angtroms, which is characteristic for globular proteins with a molecular mass of about 60 kDa. It was found that protein S1 has a tendency to form associates, and the type of the associate depends on ionic strength. These associates have, in general, two or three monomers at a moderate ionic strength, while at a low ionic strength the number of monomers exceeds three and they are packed in a compact manner. Strongly elongated associates were observed in neutron experiments at a moderate ionic strength in heavy water. The association of protein molecules was also confirmed by the data of dynamic light scattering. From these data, the translational diffusion coefficient of protein S1 at a moderate ionic strength was calculated to be (D20,w = (2.7 +/- 0.1) x 10(-7)cm2/s). This value is essentially smaller than the expected value (D20,w = (5.8 - 6.0) x 10(-7)cm2/s) for the S1 monomer in the globular conformation, indicating the association of protein molecules under equilibrium conditions.     

X-ray scattering study of hagfish protease inhibitor, a protein structurally related to complement and alpha 2-macroglobulin.

A protease inhibitor from hagfish blood plasma, homologous to human alpha 2-macroglobulin, has been studied in solution using small-angle X-ray scattering; the radius of gyration, R, was found to be 7.0 nm, the molecular weight 340,000 +/- 20,000, and the largest distance within the molecule, Dmax, 22 nm. When the inhibitor reacts with chymotrypsin, its 1:1 chymotrypsin complex is found to be more compact than the native molecule, R = 6.1 nm. A very similar conformational change is observed after the protein is reacted with methylamine. The data are consistent with models consisting of two equal elliptic cylinders with the same size as the one used as a model for the complement proteins C3 and C4 [cf. Osterberg et al. (1989) Eur. J. Biochem. 183, 507-511]. In the model for the native protein, these cylinders are arranged in an extended form, and in the one for the methylamine derivative (or chymotrypsin complex), they are closer together so that the projection of their elliptic surfaces forms an angle of about 70 degrees. These models for the hagfish protease inhibitor were expanded to models for the twice as large human alpha 2-macroglobulin using symmetry operations, and the resulting alpha 2-macroglobulin models were found to agree with those emerged from earlier studies involving electron microscopy and X-ray scattering methods.     

Structure of the proteolytic fragment F34 of calmodulin in the absence and presence of mastoparan as revealed by solution X-ray scattering.

The solution X-ray scattering technique has been applied to examine the conformations of the proteolytic fragment F34 (78Asp-148Lys) of calmodulin in the absence of both Ca2+ and mastoparan, in the presence of Ca2+ only, and in the presence of both Ca2+ and mastoparan. The radius of gyration and the molecular weight for the F34 fragment increased by 1.1 +/- 0.3 A and 19%, respectively, upon binding of both 2 mol of Ca2+/mol to the F34 fragment and mastoparan to form the tertiary complex. A smaller change was found for the Ca(2+)-saturated F34 fragment in the absence of mastoparan (0.3 +/- 0.3 A) without any change of the molecular weight. The analysis based on the small-angle scattering data showed that the F34 fragment in the presence of Ca2+ alone preserved the tertiary structure of the globular domain in the crystal to a great extent. Further analyses based on a two-domain model showed that the center-to-center distance between F34 and mastoparan is about 12.7 A, if the structure of the F34 fragment in the presence of mastoparan resembles that in the absence of mastoparan and if mastoparan in the complex retains an alpha-helical conformation. The modeling studies using their crystal structure coordinates have been made on the basis of the solution X-ray scattering data. The combined results support a model proposed by Persechini and Kretsinger [Persechini, A., & Kretsinger, R. H. (1988) J. Cardiovasc. Pharmacol. 12 (Suppl. 5), S1-S12], although the center-to-center distance between mastoparan and the F34 fragment is shorter by about 5 A than that in their model.(ABSTRACT TRUNCATED AT 250 WORDS)     

Yeast hexokinase in solution exhibits a large conformational change upon binding glucose or glucose 6-phosphate.

Using small-angle X-ray scattering from solutions of yeast hexokinase, we have measured the radii of gyration of the monomeric B isozyme and its complexes with sugar substrates. We find that the radius of gyration decreases by 0.95 +/- 0.24 A upon binding glucose and 1.25 +/- 0.28 A upon binding glucose 6-phosphate. This observed reduction in radius of gyration in the presence of glucose is the same as that calculated from the coordinates of the high-resolution crystal structures of native hexokinase B and a glucose complex with hexokinase A. Thus, these measurements suggest that the dramatic closing of the slit between the two lobes of hexokinase observed in the crystal structures (Bennett, W.S., & Steitz, T.A. (1978) Proc. Natl. Acad. Sci. U.S.A. 75, 4848--4852) occurs in solution when either glucose or glucose 6-phosphate is bound.     

Conformation of human IgG subclasses in solution. Small-angle X-ray scattering and hydrodynamic studies

The structure of six human myeloma proteins: IgG1(Bal), IgG2(Klu), IgG3(Bak), IgG3(Het), IgG4(Kov) and IgG4(Pol), was studied in solution using small-angle X-ray scattering and hydrodynamic methods. For IgG1(Bal) and IgG3(Het) the experimental data, including radius of gyration (Rg degree), radii of gyration of the cross-section (Rq1, Rq2), intrinsic viscosity [eta], sedimentation coefficient (S degree 20,w) and molecular mass, were interpreted in terms of structural models based on the Fab and Fc conformations, observed in crystal, by varying the relative positions of the Fab and Fc parts, i.e. their relative angles and distances. The values Rg degree = (6.00 +/- 0.05) nm, S degree 20,w = (6.81 +/- 0.10) S and [eta] = 0.0062 +/- 0.0005 cm3/mg obtained for IgG1(Bal) are compatible with a planar model in which the angle between the Fab arms is about 120 degrees. For IgG3(Het) the following data were obtained: Rg degree = (4.90 +/- 0.05) nm, S degree 20,w = (6.32 +/- 0.01) S and [eta] = (0.0065 +/- 0.0005) cm3/mg. The apparent contradiction between the higher molecular mass and lower Rg degree and S degree 20,w values for IgG3(Het) in comparison to IgG1(Bal) can be resolved by proposing a 'non-planar' (tetrahedral) molecular shape, in which the long hinge peptide is in a folded conformation and the two Fab and Fc parts are in a closely packed arrangement. In this model the angle between the two Fab arms is about 90 degrees, in the average position. The X-ray scattering and hydrodynamic behaviour of the IgG2 and IgG4 types of antibodies appeared to be similar to IgG1(Bal). The parameters of the two IgG3 proteins are similar while they are different to the others.     

Glutathione conjugation and pharmacokinetics of 2-bromo-3-phenylpropionic acid in vitro and in the rat in vivo.

Glutathione (GSH) conjugation of the chiral compound 2-bromo-3-phenylpropionic acid (BPP) was studied in vitro and in the rat in vivo. GSH conjugation of BPP, catalyzed by a mixture of glutathione-S-transferases (GST's) from rat liver cytosol in vitro, was stereoselective: at a substrate concentration of 250 microM, (R)-BPP was more rapidly conjugated than (S)-BPP (R/S-ratio = 2.6). The blood elimination kinetics of the separate BPP enantiomers and the biliary excretion kinetics of the corresponding GSH conjugates were studied in the rat in vivo after administration of (R)- or (S)-BPP at a dose level of 50 mumol/kg. Elimination of (R)-BPP from blood was faster than that of (S)-BPP: half lives were 9 +/- 2 min for (R)-BPP and 13 +/- 1 min for (S)-BPP. The biliary excretion rate of the GSH conjugate of (R)-BPP declined monoexponentially, while that of the GSH conjugate of (S)-BPP displayed a biphasic profile. Half lives of excretion were 13 +/- 1 for the GSH conjugate of (R)-BPP, and 11 +/- 2 for the GSH conjugate of (S)-BPP (second phase). The first phase in the biliary excretion of the GSH conjugate of (S)-BPP could not be attributed to capacity limitation of biliary transport carriers as higher excretion rates were attained upon administration of higher doses (100 and 200 mumol/kg) of (S)-BPP). The blood elimination profiles of (R)- and (S)-BPP differed greatly from the biliary excretion profiles of the corresponding GSH conjugates. This suggests that the kinetics of BPP conjugate excretion are determined by other processes than hepatic GSH conjugation.     

The conformation of a large RNA fragment from the E.coli ribosomal 16S-RNA. An X-ray and neutron small-angle scattering study.

A large 12S RNA fragment which constitutes the 5' two-thirds of 16S-RNA from the E. coli 30S subunit has been investigated by small-angle X-ray and neutron scattering. The results indicate that in reconstitution buffer the 12S-RNA fragment has a molecular weight of 270,000 +/- 20,000 and a radius of gyration of 7.1 nm. The scattering data are compatible with the RNA being folded into two major domains with the shapes of two adjacent, quite similar cylinders.     

Conformation of poly(methacrylic acid) in acidic aqueous solution studied by small angle X-ray scattering

The nature of the contracted form of poly(methacrylic acid) PMA chain in salt-free acidic aqueous solution was studied by analyzing scattering curves registered by small-angle X-ray scattering, comparing it with those of PMA in methanol at 26 degrees C and of partially neutralized PMA in aqueous solution containing added salt (the concentration of added salt, Cs=0.1 M NaF). It is shown that the distribution of segments in the contracted form as well as that of PMA in methanol is that of a random-coil in a theta medium and that this distribution of segments is stable over a fair range of degrees of ionization alpha for Cs below 0.1 M. Moreover, the persistence length of PMA at Cs=0.1 M (4+/-0.5 A) is substantially constant throughout the entire range of alpha, indicating that the contracted form of PMA changes to an expanded random-coil in a higher pH region without a significant change in the chain flexibility.     

SARS CoV main proteinase: The monomer-dimer equilibrium dissociation constant

The SARS coronavirus main proteinase (SARS CoV main proteinase) is required for the replication of the severe acute respiratory syndrome coronavirus (SARS CoV), the virus that causes SARS. One function of the enzyme is to process viral polyproteins. The active form of the SARS CoV main proteinase is a homodimer. In the literature, estimates of the monomer-dimer equilibrium dissociation constant, KD, have varied more than 65,0000-fold, from <1 nM to more than 200 microM. Because of these discrepancies and because compounds that interfere with activation of the enzyme by dimerization may be potential antiviral agents, we investigated the monomer-dimer equilibrium by three different techniques: small-angle X-ray scattering, chemical cross-linking, and enzyme kinetics. Analysis of small-angle X-ray scattering data from a series of measurements at different SARS CoV main proteinase concentrations yielded KD values of 5.8 +/- 0.8 microM (obtained from the entire scattering curve), 6.5 +/- 2.2 microM (obtained from the radii of gyration), and 6.8 +/- 1.5 microM (obtained from the forward scattering). The KD from chemical cross-linking was 12.7 +/- 1.1 microM, and from enzyme kinetics, it was 5.2 +/- 0.4 microM. While each of these three techniques can present different, potential limitations, they all yielded similar KD values.     

Dimer formation of subunit G of the yeast V-ATPase

The G subunit of the vacuolar ATPase (V-ATPase) is a component of the stalk connecting the V(1) and V(O) sectors of the enzyme and is essential for normal assembly and function. Subunit G (Vma10p) of the yeast V-ATPase was expressed in Escherichia coli as a soluble protein and was purified to homogeneity. The molecular mass of subunit G, determined by Native-polyacrylamide gel electrophoresis, gel filtration analysis and small-angle X-ray scattering, was approximately 28+/-2 kDa, indicating that this protein is dimeric. With a radius of gyration (R(g)) and a maximum size (D(max)) of 2.7+/-0.2 nm and 8.0+/-0.3 nm, respectively, the G-dimer is rather elongated. To understand which region of subunit G is required to mediate dimerization, a G(38-144) form (the carboxyl-terminus) was expressed and purified. G(38-144) is homogeneous, with a molecular mass of approximately 12+/-3 kDa, indicating a monomeric form in solution.     

Effect of cholesterol on the bilayer thickness in unilamellar extruded DLPC and DOPC liposomes: SANS contrast variation study

Small-angle neutron scattering on extruded unilamellar vesicles in water was used to study bilayer thickness when cholesterol (CHOL) was added to dilauroylphosphatidylcholine (DLPC) and dioleoylphosphatidylcholine (DOPC) bilayers in molar fraction 0.44. Using the H2O/2H2O contrast variation and the small-angle form of Kratky-Porod approximation, the bilayer gyration radius at infinite contrast R(g,infinity) and the bilayer thickness parameter d(g,infinity) = 12(0.5)R(g,infinity) were obtained at 25 degrees C. Addition of CHOL to DLPC increased the d(g,infinity) from 4.058 +/- 0.028 nm to 4.62 +/- 0.114 nm, while in case of DOPC the d(g,infinity) values were the same in the absence (4.618 +/- 0.148 nm) and in the presence (4.577 +/- 0.144 nm) of CHOL within experimental errors. The role of CHOL-induced changes of bilayer thickness in the protein insertion, orientation and function in membranes is discussed.     

Small-angle neutron scattering study of the lipid bilayer thickness in unilamellar dioleoylphosphatidylcholine vesicles prepared by the cholate dilution method: n-decane effect

Previous X-ray diffraction studies on fully hydrated fluid lamellar egg phosphatidylcholine phases indicated a approximately 10 A increase of bilayer thickness in the presence of excess n-decane [Biochim. Biophys. Acta 597 (1980) 455], while the small-angle neutron scattering (SANS) on unilamellar extruded dioleoylphosphatidylcholine (DOPC) vesicles detected substantially smaller 2.4+/-1.3 A bilayer thickness increase at n-decane/DOPC molar ratio of 1.2 [Biophys. Chem. 88 (2000) 165]. The purpose of the present study is to investigate the n-decane effect on the bilayer thickness in unilamellar DOPC vesicles prepared by the sodium cholate (NaChol) dilution method. Mixed DOPC+NaChol micelles at DOPC and NaChol concentrations of 0.1 mol/l were prepared in 2H(2)O containing 0.135 mol/l NaCl. This micellar solution was diluted in 0.135 mol/l NaCl in 2H(2)O to reach the final DOPC and NaChol concentrations of 0.008 mol/l. Thirty microliters of n-decane solution in methanol was added to 1 ml of this dispersion. After methanol evaporation, SANS was conducted on the dispersions. From the Kratky-Porod plot ln[I(Q)Q(2)] vs. Q(2) of SANS intensity I(Q) in the range of scattering vector values Q corresponding to interval 0.001 A(-2)相似文献   

Validity/reliability of sweat analysis by whole-body washdown vs. regional collections

Six subjects (25.3 +/- 3.3 yr, mean +/- SD) exercised for 60 min at 42 +/- 4 [low (L)], 55 +/- 6 [moderate (M)], and 67 +/- 4 %VO2max [high (H)] in a moderate environment. Sweat collected from upper back (UB), lower back (LB), midchest (MC), stomach (S), and thigh (T) areas as well as by whole-body washdown (W) was analyzed for urea nitrogen (N). With the exception of the L where all regional measures were similar, all sites overestimated W (several significantly, P less than 0.05). Regression analysis estimations of W (mg/h) from regional collections were as follows--L: W = 0.727 (S) - 1.366(UB) + 1.181(T) + 65.470 +/- 29.5, R = 0.90; M: W = 0.598(MC) - 0.649(UB) + 0.244(LB) + 43.238 +/- 30.4, R = 0.99; H: W = 0.274(S) - 0.560(T) + 0.223(MC) + 131.104 +/- 4.3, R = 0.99; All Intensities: W = 0.497(MC) - 0.483(T) + 0.112(LB) + 69.554 +/- 31.5, R = 0.96. W recovery of exogenous urea N applied to each subject's body was 98.3 +/- 2.7% (mean +/- SE). Interinvestigator reliability coefficient (r = 0.511) was significant (P less than 0.01) but relatively low and the between investigator urea N recovery (93.3 +/- 3.7 vs. 103.2 +/- 3.5%) was significantly different (P less than 0.05). Repeated W determinations by the same investigator were not different (P greater than 0.05), but intrainvestigator reliability coefficients differed widely (0.385 vs. 0.820). Together, these data indicate that W solute recovery can be high; however, both inter- and intrainvestigator reliability can vary.(ABSTRACT TRUNCATED AT 250 WORDS)     

X-ray scattering of antibodies: the monomeric 8S subunit of human IgM

8S monomeric subunit of a human immunoglobulin M was investigated by small-angle X-ray scattering. The following molecular parameters were determined: radius of gyration 5.9 nm, maximum length 21 nm, hydrated volume 410 nm³ and two radii of gyration of the cross-section: 2.6 and 1.8 nm. A model equivalent in scattering was found and compared with the model for a human IgG also based on small-angle X-ray scattering data. The Cμ2 domain of the IgM obviously has a very loose structure, and the Fab angle of the 8S IgM (90°) is smaller than that of the IgG (134°).     

Molecular and complex-chemical studies on methemoglobin from Chironomus thummi thummi and various isolated fractions]

1. Six different hemoglobin (Hb) fractions were isolated and characterized from the larvae of Chironomus thummi thummi using column chromatographic procedures. 2. Chromatographic and sedimentation-analytic studies (sedimentation coefficients of 2.0 +/- 0.2 (S)) have shown three Hb fractions to exist basically in a monomeric form. The molecular weight of component M-2 was determined by sedimentation equilibrium technique to be 15,470 +/- 400. The dimeric Hb was found to have sedimentation coefficients of 3.0 +/- 0.1 (S) in the weakly acidic pH region. In alkaline milieu, the reversible dissociation proceeds into the monomeric molecules (S20, W = 1.9 +/- 0.1 (S)). Molecular weights vary between pH 5.7 and 9.8 not only with hydrogen ion concentration, but also with protein concentration in correspondence with a dissociation-association equilibrium consisting of monomers and dimers. 3. For the Hb fraction M-2, a friction ratio of f/fo = 1.03 was calculated, suggesting an almost spherical shape of this protein. In contrast, the dimeric component appears to have a much more asymmetric structure (f/fo = 1.19). 4. The indivdual MetHb fractions bind the ligands: fluoride, imidazole and azide with different affinities.     

Analysis of collagen fibril diameter distribution in connective tissues using small-angle X-ray scattering

Analysis of the diameters of collagen fibrils provides insight into the structure and physical processes occurring in the tissue. This paper describes a method for analyzing the frequency distribution of the diameters of collagen fibrils from small-angle X-ray scattering (SAXS) patterns. Frequency values of fibril diameters were input into a mathematical model of the form factor to calculate the equatorial intensity which best fits the experimentally derived data from SAXS patterns. A minimization algorithm utilizing simulated annealing (SA) was used in the fitting procedure. The SA algorithm allowed for random sampling of the frequency values, and was run iteratively to build up an optimized frequency distribution of fibril diameters. Results were obtained for collagen samples from sheep spine ligaments. The mean fibril diameter value obtained from this data-fitting method was 73 nm+/-20 nm (S.D.). From scanning transmission electron microscopy, the mean diameter was found to be 69 nm+/-14 nm (S.D.). The good agreement between the two methods demonstrates the reliability of the SAXS method for the tissue examined. The non-destructive nature of this technique, as well as its statistical robusticity and capacity for large sampling, means that this method is both quick and effective.     

Shape, symmetry, hydration and secondary structure of the legumin from Vicia faba in solution

The following physical parameters of the legumin from Vicia faba were determined by means of small-angle X-ray scattering, quasi-elastic light scattering and circular dichroism: molar mass, M = 3.5 X 10(5) g/mol; radius of gyration, Rg = 4.45 nm; maximum dimension, L = 13 nm; translational diffusion coefficient, D0(20),w = 3.38 X 10(-7) cm2 X s-1; alpha-helix content about 15%; content of beta-sheets 10%; dihedral point group symmetry of the molecule 32.