Sedimentation Coefficient S020 Research Articles

The solution structure of the unbound IgG Fc receptor CD64 resembles its crystal structure: Implications for function.

FcγRI (CD64) is the only high-affinity Fcγ receptor found on monocytes, macrophages, eosinophils, neutrophils and dendritic cells. It binds immunoglobulin G (IgG) antibody-antigen complexes at its Fc region to trigger key immune responses. CD64 contains three immunoglobulin-fold extracellular domains (D1, D2 and D3) and a membrane-spanning region. Despite the importance of CD64, no solution structure for this is known to date. To investigate this, we used analytical ultracentrifugation, small-angle X-ray scattering, and atomistic modelling. Analytical ultracentrifugation revealed that CD64 was monomeric with a sedimentation coefficient s020,w of 2.53 S, together with some dimer. Small-angle X-ray scattering showed that its radius of gyration RG was 3.3-3.4 nm and increased at higher concentrations to indicate low dimerization. Monte Carlo modelling implemented in the SASSIE-web package generated 279,162 physically-realistic trial CD64 structures. From these, the scattering best-fit models at the lowest measured concentrations that minimised dimers revealed that the D1, D2 and D3 domains were structurally similar to those seen in three CD64 crystal structures, but showed previously unreported flexibility between D1, D2 and D3. Despite the limitations of the scattering data, the superimposition of the CD64 solution structures onto crystal structures of the IgG Fc-CD64 complex showed that the CD64 domains do not sterically clash with the IgG Fc region, i.e. the solution structure of CD64 was sufficiently compact to allow IgG to bind to its high-affinity Fcγ receptor. This improved understanding may result in novel approaches to inhibit CD64 function, and opens the way for the solution study of the full-length CD64-IgG complex.

Solution structures of human myeloma IgG3 antibody reveal extended Fab and Fc regions relative to the other IgG subclasses

Human immunoglobulin G subclass 3 (IgG3) possesses a uniquely long hinge region that separates its Fab antigen-binding and Fc receptor-binding regions. Owing to this hinge length, the molecular structure of full-length IgG3 remains elusive, and the role of the two conserved Fc glycosylation sites are unknown. To address these issues, we subjected glycosylated and deglycosylated human myeloma IgG3 to multidisciplinary solution structure studies. Using analytical ultracentrifugation, the elongated structure of IgG3 was determined from the reduced sedimentation coefficients s020,w of 5.82 to 6.29 S for both glycosylated and deglycosylated IgG3. X-ray and neutron scattering showed that the Guinier RG values were 6.95 nm for glycosylated IgG3 and were unchanged after deglycosylation, again indicating an elongated structure. The distance distribution function P(r) showed a maximum length of 25 to 28 nm and three distinct maxima. The molecular structure of IgG3 was determined using atomistic modeling based on molecular dynamics simulations of the IgG3 hinge and Monte Carlo simulations to identify physically realistic arrangements of the Fab and Fc regions. This resulted in libraries containing 135,135 and 73,905 glycosylated and deglycosylated IgG3 structures, respectively. Comparisons with the X-ray and neutron scattering curves gave 100 best-fit models for each form of IgG3 that accounted for the experimental scattering curves. These models revealed the first molecular structures for full-length IgG3. The structures exhibited relatively restricted Fab and Fc conformations joined by an extended semirigid hinge, which explains the potent effector functions of IgG3 relative to the other subclasses IgG1, IgG2, and IgG4.

Solution structure of deglycosylated human IgG1 shows the role of CH2 glycans in its conformation

The human immunoglobulin G (IgG) class is the most prevalent antibody in serum, with the IgG1 subclass being the most abundant. IgG1 is composed of two Fab regions connected to a Fc region through a 15-residue hinge peptide. Two glycan chains are conserved in the Fc region in IgG; however, their importance for the structure of intact IgG1 has remained unclear. Here, we subjected glycosylated and deglycosylated monoclonal human IgG1 (designated as A33) to a comparative multidisciplinary structural study of both forms. After deglycosylation using peptide:N-glycosidase F, analytical ultracentrifugation showed that IgG1 remained monomeric and the sedimentation coefficients s020,w of IgG1 decreased from 6.45 S by 0.16–0.27 S. This change was attributed to the reduction in mass after glycan removal. X-ray and neutron scattering revealed changes in the Guinier structural parameters after deglycosylation. Although the radius of gyration (RG) was unchanged, the cross-sectional radius of gyration (RXS-1) increased by 0.1 nm, and the commonly occurring distance peak M2 of the distance distribution curve P(r) increased by 0.4 nm. These changes revealed that the Fab-Fc separation in IgG1 was perturbed after deglycosylation. To explain these changes, atomistic scattering modeling based on Monte Carlo simulations resulted in 123,284 and 119,191 trial structures for glycosylated and deglycosylated IgG1 respectively. From these, 100 x-ray and neutron best-fit models were determined. For these, principal component analyses identified five groups of structural conformations that were different for glycosylated and deglycosylated IgG1. The Fc region in glycosylated IgG1 showed a restricted range of conformations relative to the Fab regions, whereas the Fc region in deglycosylated IgG1 showed a broader conformational spectrum. These more variable Fc conformations account for the loss of binding to the Fcγ receptor in deglycosylated IgG1.

Structure and mucoadhesion of mussel glue protein in dilute solution.

Purified mussel adhesive protein mefp-1 (Mytilus edulis foot protein 1) has been studied regarding its state of oligomerization and gross conformation in dilute solution. Sedimentation equilibrium in the analytical ultracentrifuge of a dilute solution of protein (0.4 mg/mL) in acetate buffer at pH 4.5 and I = 0.10 M yielded an apparent molecular weight (whole distribution weight average, Mw, app) of 114 000 +/- 5000 via the "M" procedure, a value in almost exact agreement with the monomeric molecular weight obtained by MALDI mass spectrometry. At this low concentration, it is reasonable to assume thermodynamic ideality, i.e., Mw,app approximately Mw. This result, together with plots of point weight average apparent molecular weight versus concentration for three different loading concentrations (0.4, 0.8, 1.0 mg/mL), clearly demonstrates that this protein is essentially monomeric in dilute solution. Sedimentation velocity experiments yielded an estimate of the sedimentation coefficient s020,w = 2.34 +/- 0.17 S, which for M = 110 000 gives a frictional ratio f/f0 = 3.2 +/- 0.3. The interpretation of this, in terms of an extended rather than globular conformation for the structure of mefp-1 in dilute solution, is considered, within plausible limits of molecular hydration, and models for the structure in solution are considered, in light of the thermodynamic nonideality behavior of these molecules and previously published circular dichroism data. The significance of these observations in terms of the bioadhesive properties of mefp-1 is described, and the very strong interaction in dilute solution with a mucin glycoprotein is demonstrated.

Size and conformation ofArtemiaribosomal RNA's, free in solution

AbstractThe conformation and structure of the small and large ribosomal subunits, as well as their interaction, is influenced by the conformation, structure and interactions of the ribosomal RNA's. Indeed, the structure of the ribosomal particles, as well as of their rRNA's in solution, depends heavily on the Mg2+ and K+ concentrations (e.g. Elson Spitnik-Elson, 1981). Further, the interaction between both subunits in the ribosome and between the rRNA's free in solution on the other hand is only possible in a limited and similar range of Mg2+ concentrations (Burma et al., 1983). Therefore, we have followed up a study of the physical properties of eukaryotic ribosomal particles with a study of the size and conformation of their rRNA's free in solution. Information about the structure of the rRNA was obtained by measuring their sedimentation and diffusion coefficient. The main experimental emphasis has been put on the determination of the sedimentation coefficients s020,w of the rRNA's at Mg2+ concentration...

Physical characteristics of 16 S rRNA under reconstitution conditions.

The hydrodynamic shape and conformation of the 16 S ribosomal RNA in reconstitution buffer at both 4 degrees C and 37 degrees C were determined and compared with the corresponding properties of the 30 S ribosomal subunit at 4 degrees C in order to understand the role of the RNA molecule in the assembly of the 30 S subunit. At 4 degrees C, the 16 S rRNA has a sedimentation coefficient s020,w of 21.0 S, a diffusion coefficient D020,w of 1.72 X 10(-7) cm2/s, a frictional coefficient f/fmin of 2.37, and a hydrodynamic radius of 125 A. At 37 degrees C, the 16 S rRNA has a sedimentation coefficient s020,w of 18.4 S, a diffusion coefficient D020,w of 1.39 X 10(-7) cm2/s, a frictional coefficient f/fmin of 2.91, and a hydrodynamic radius of 153 A. At 4 degrees C, the 30 S subunit has a sedimentation coefficient s020,w of 31.8 S, a diffusion coefficient D020,w of 1.97 X 10(-7) cm2/s, a frictional coefficient f/fmin of 1.77, and a hydrodynamic radius of 109 A. These results suggested that the free RNA in solution at 4 degrees C is less folded than the RNA in the ribosomal subunit. At 37 degrees C, the free 16 S rRNA is unfolded when compared to the structure of the same RNA at 4 degrees C. This implies that the folding step accompanying the RI to RI transformation in the assembly process needs the presence of both the RNA and core proteins.

The Structure of Acid-soluble Basement Membrane Collagen from Bovine Anterior Lens Capsule: Molecular Parameters and Thermal Gelation Properties

Basement membrane collagen was extracted from bovine anterior lens capsules with acetic acid in the presence of protease and proteinase inhibitors. This undegraded soluble basement membrane has a sedimentation coefficient S020,W = 7.8 × 10-13 sec-1, a diffusion coefficient D020,w = 5.82 × 10-8 cm2/sec and a zero-shear intrinsic viscosity [***1)] = 2200 cm3/g. These data lead to a molecular weight, Mr of 1.1 × 106. Electron microscopic observation indicates that the molecules are rod-like in character with a length of 300 nm. Theoretical models describing the intrinsic viscosity in terms of the distribution of mass in such an elongated molecule show that a dumbbell-like structure is the only tenable model. In this model a single 300 nm rod-like region joins two more densely packed globular regions. The globular domains contain hydroxyproline and may have collagenase sensitive regions since digestion with purified bacterial collagenase at 37°C for 18 h degrades these domains as well as the central triple-helical domain. The intact acid-soluble basement membrane collagen precipitates from neutral pH solutions upon warming 4°C stable solutions. The pH, ionic strength and concentration dependence parameters of this thermal precipitation have been examined. The basement membrane collagen precipitation appears to be dominated by endregion domain hydrophobic interactions. The end-regions are required for precipitation in contrast to the Type I pro collagen situation wherein the intact end-regions inhibit thermal gelation. Electron microscopic examination of the basement membrane thermal gels shows networks of very thin filaments which may be related directly to the structure within intact basement membrane.

Isolation and partial characterization of proteins from pea (Pisum sativum L.)

AbstractPea flour with a protein content (Nx6,25) of 25% was subjected to an extraction by 10% NaCl. The changes of the meal particles during the extraction were studied by electron microscopy. 89% of the nitrogen substances were extracted, 14% of which are albuminsThe nitrogen solubility curve of the meal has a minimum of 18% solubility at pH 4‐5The proteins in the NaCl extract were fractionated using dialysis (separation of globulins and albumins) and preparative gel chromatography (separation of the 11 S and 7 S globulins). The 11 S and 7 S globulins were homogenous in gel chromatography and ultracentrifugation. Their sedimentation coefficients s020 w were 11,9 S and 6,4 S, respectively. They differ significantly in the gel electrophoresisThe amino acid composition of the isolated globulins shows a relatively high content of glutamin and aspartic acid as well as arginine and a lack of sulphur containing amino acids which are limiting. EAA indices of 69, 63, 62, 32 and 78 were calculated for pea flour, the raw globulin fraction, the 11 S and 7 S fraction, and the albumine fraction, respectively. Therefore for preserving the nutritive value of the pea material preparation processes are to be applied which exclude the loss of albumins during the protein isolation.

Characterization of the histone core complex.

A stable histone core complex containing equimolar ratios of H2A, H2B, H3, and H4 has been isolated from chicken erythrocyte chromatin in high salt. This complex has been characterized by sedimentation and chemical cross-linking studies. In velocity ultracentrifugation, only one single sharp sedimentation boundary has been observed with sedimentation coefficient S020,w = 3.8 +/- 0.1. The molecular weight of the histone core complex has been investigated by low-speed sedimentation equilibrium studies over a wide range of protein concentration. Analysis of the apparent weight-average molecular weight as a function of concentration indicates that the histone core complex in 2 M NaCl, pH 9.0, is in equilibrium between a tetramer (H2A)(H2B)(H3)(H4) and an octamer [(H2A)(H2B)(H3)(H4)]2 species with a tetramer molecular weight of 55,000. The equilibrium constant K is approximately 1.2 X 10(-5) liter per mol at 10 degrees. Evidence of such a tetramer-octamer equilibrium in solution is also supported by the results of our chemical crosslinking experiments on the histone core complex.

Isolation and characterization of two beta1-glycoproteins of human serum.

Two immunoelectrophoretically defined, heretofore unidentified beta(1)-globulins of human serum, provisionally designated beta(1C)- and beta(1A)-globulin, were isolated by means of preparative electrophoresis and chromatography on anion exchange cellulose. The sedimentation coefficient S(0) (20, w) of beta(1C)-globulin was shown to be 9.5 S, and that of beta(1A)-globulin, 6.9 S. Both proteins were found to contain similar amounts of carbohydrate, to be devoid of lipids, and to possess the solubility characteristics of euglobulins. In the Ouchterlony double diffusion test they gave the reaction of partial identity, which revealed beta(1A)-globulin to be anti-genically deficient as compared to beta(1C)-globulin. beta(1A)-globulin could not be detected in fresh sera and beta(1C)-globulin was absent from aged sera. Highly purified beta(1C)-globulin stored at 1 degrees C. was converted to beta(1A)-globulin within 4 to 6 weeks, and at 37 degrees C. was converted within 6 days. The likelihood of a dimer-monomer relationship between these two proteins was discussed.