Structure and flexibility of individual immunoglobulin G molecules in solution

In contrast to averaging methods of determining structure, such as X-ray diffraction, NMR, and single-particle tomography, cryo-electron tomography allows three-dimensional imaging of an individual object in solution. The method has previously been used to study cells and very large macromolecules. We have used cryo-electron tomography to analyze a monoclonal IgG, with a molecular weight of only 150 kDa. Tomograms reveal y-shaped IgG molecules with three protruding subunits. Docking X-ray structures enabled us to recognize the three subunits as two ellipsoidal Fab arms and a heart-shaped Fc stem. Each subunit has a similar structure in the tomograms and in the X-ray map. Notably, the positions of the Fab arms relative to the Fc stem differed greatly from one molecule to another. The large flexibility of IgG in solution is most likely of functional significance in antigen recognition. This distribution of individual structures provides a qualitative insight into the system dynamics.     

Opposite effect of albumin on the erythrocyte aggregation induced by immunoglobulin G and fibrinogen

The effect of albumin on the immunoglobulin G (IgG)-induced and fibrinogen-induced aggregation of human erythrocytes was quantitatively examined by using a rheoscope combined with a television image analyzer and a computer. As albumin concentration in the medium was increased, the IgG-induced erythrocyte aggregation was inhibited, while the fibrinogen-induced erythrocyte aggregation was accelerated (albumin itself was not able to aggregate erythrocytes). These relations were empirically expressed by the equations, v = aG1.8/A and v = a'F1.5 (A + b'), respectively (v, the velocity of erythrocyte aggregation; A, G and F, the concentrations of albumin, IgG and fibrinogen, respectively; a, a' and b', constant). The IgG-induced erythrocyte aggregation was remarkably inhibited by the addition of poly(glutamic acid), but the fibrinogen-induced erythrocyte aggregation was not. A mechanism for the interaction of immunoglobulin G and fibrinogen with the surface of erythrocytes was proposed.     

A new approach to the depletion of albumin and immunoglobulin G from human serum

The use of proteomic analysis to find potential diagnostic biomarkers is limited by the presence of serum albumin (HSA) and immunoglobulin (IgG) at high concentrations in patients’ blood; these substances impede the detection of serum proteins with similar molecular weights. Recombinant HSA- and IgG-binding polypeptides are used as ligands in creating sorbents for complete removal of the proteins by affinity chromatography. The binding specificity of the sorbents for HSA and IgG is higher than that of the conventionally used antibodies. A composite sorbent enabling the depletion of HSA and IgG from serum by single-step affinity chromatography was obtained. The developed sorbents were used to prepare serum for proteomic analysis.     

Failure of selenomethionine residues in albumin and immunoglobulin G to protect against peroxynitrite.

Selenomethionine has been suggested to protect against peroxynitrite by quenching it in vivo. Selenomethionine is distributed randomly in the methionine pool. Albumin and IgG were purified from plasma of a human being before and after 28 days of supplementation with 400 microg selenium/day as selenomethionine. The albumin contained 1 selenium atom, presumably as selenomethionine, per 8000 methionine residues before supplementation and 1 per 2800 after supplementation. Although this ratio suggested that selenomethionine would not have as great an effect in quenching peroxynitrite as would methionine, direct testing of the albumin and IgG fractions was carried out to assess the ability of these proteins to prevent peroxynitrite oxidation of dihydrorhodamine 123 to rhodamine 123. The ability of the albumin preparations to resist nitration of tyrosine residues was also assessed. The high-selenomethionine preparations of the proteins had no greater effect in quenching the peroxynitrite than did the normal-selenomethionine preparations. These results do not support the proposal that selenomethionine in proteins contributes to in vivo protection against peroxynitrite.     

Characterization of the photoproducts of protoporphyrin IX bound to human serum albumin and immunoglobulin G

Clinically useful photosensitisers (PSs) are likely bound to subcellular and molecular targets during phototherapy. Binding to a macromolecule has the potential to change the photophysical and photochemical characteristics of the PSs that are crucial for their phototoxicity and cell-killing activity. We investigated the effects of binding of a specific PS (protoporphyrin IX or PPIX) to two proteins, human serum albumin (HSA) and a commercially available immunoglobulin (IgG). These two proteins provide two different environments for PPIX. The albumin binds PPIX in hydrophobic binding sites located in subdomain IIA and IIIA, conversely IgG leaves PPIX exposed to the solvent. We show that photophysical parameters such as emission maxima and fluorescence lifetime depend on the binding site. Our results indicate that the different binding site yields very different rates of formation of photoproducts (more than three times higher for PPIX bound to HSA than to IgG) and that different mechanisms of formation may be occurring. Our characterization shows the relevance of protein binding for the photochemistry and ultimately the phototoxicity of PSs.     

Quantitation of human immunoglobulin G and albumin in electroimmunodiffusion gels containing ionic and nonionic detergents

Quantitation of human immunoglobulin G (IgG) and albumin by agarose electroimmunodiffusion is influenced by the incorporation of ionic and nonionic detergents in the gel. The highest concentrations of each detergent at which human IgG and albumin determinations could be performed without perturbing the quantitations were 4% Triton X-100, 4% Tween 80, 1% NP-40, 0.5% sodium deoxycholate (SDOC), 0.5% Zwittergent, and 0.1% sodium dodecyl sulfate (SDS), and mixtures of Triton X-100, SDOC, and SDS. These detergent combinations all resulted in greater perturbations of albumin quantitation than of IgG. Immunoprecipitation of human IgG was quantitated in the absence and presence of Triton X-100, Zwittergent, and SDS. SDS was shown to cause nonspecific precipitation, whereas below 1% Triton X-100 or 0.5% Zwittergent no effects upon the immunoprecipitations were observed.     

Hydrogen bonding and equilibrium protium-deuterium fractionation factors in the immunoglobulin G binding domain of protein G

Protium-deuterium fractionation factors (phi) were determined for more than 85% of the backbone amide protons in the IgG binding domains of protein G, GB1 and GB2, from NMR spectra recorded over a range of H2O/D2O solvent ratios. Previous studies suggest a correlation between phi and hydrogen bond strength; amide and hydroxyl groups in strong hydrogen bonds accumulate protium (phi < 1), while weak hydrogen bonds accumulate deuterium (phi > 1). Our results show that the alpha-helical residues have slightly lower phi values (1.03 +/- 0.05) than beta-sheet residues (1.12 +/- 0.07), on average. The lowest phi value obtained (0.65) does not involve a backbone amide but rather is for the interaction between two side chains, Y45 and D47. Fractionation factors for solvent-exposed residues are between the alpha-helix and beta-sheet values, on average, and are close to those for random coil peptides. Further, the difference in phiav between alpha-helix and solvent-exposed residues is small, suggesting that differences in hydrogen bond strength for intrachain hydrogen bonds and amide...water hydrogen bonds are also small. Overall, the enrichment for deuterium suggests that most backbone...backbone hydrogen bonds are weak.     

Localization of bound water in the solution structure of the immunoglobulin binding domain of streptococcal protein G. Evidence for solvent-induced helical distortion in solution.

The presence of bound water in the solution structure of the IgG binding domain of streptococcal protein G has been investigated by nuclear magnetic resonance using three-dimensional 1H rotating frame Overhauser 1H-15N multiple quantum coherence spectroscopy. The backbone amide protons of three residues, Ala20, Gln32 and Tyr33, are found to be in close proximity to bound water. Examination of the three-dimensional structure of the IgG binding domain indicates that in the vicinity of these three residues there are no backbone groups that do not already participate in hydrogen bonding and there are no suitably placed side-chain groups available for hydrogen bonding with water. As the lifetime of the bound water detected in this nuclear magnetic resonance experiment is greater than about one nanosecond, it is likely that the two bound water molecules participate in a bifurcating hydrogen bonding network comprising a CO-NH hydrogen bonded pair, such that the water molecule accepts a hydrogen bond from the NH proton and donates one to the carbonyl oxygen with the result that the amide proton is involved in a three center hydrogen bond. On the basis of the structure, one water molecule participates in such an interaction with the Ala20(NH)-Met1(CO) hydrogen bonded pair at the beginning of an anti-parallel beta-sheet, and the other with the Tyr33(NH)-Val29(CO) hydrogen bonded pair in the single alpha-helix. The latter, which is external and solvent accessible, is associated with a distortion in the alpha-helix centered around Tyr33 which consists of a significant increase in the CO(i-4)-N(i) and CO(i-4)-NH(i) distances relative to those in the rest of the helix, as well as a significant departure in the phi, psi angles of Tyr33 relative to regular helical geometry. Such solvent induced distortions in alpha-helices have been previously noticed in crystal structures and were postulated as possible folding intermediates for helical structures. The present observation of this phenomenon in solution indicates, however, that these water molecules are tightly bound and represent an integral part of the protein framework.     

Light resonance energy transfer-based methods in the study of G protein-coupled receptor oligomerization

Since most of the functions in cells are mediated by multimeric protein complexes, the determination of protein-protein interactions is an important step in the study of cellular mechanisms. Traditionally, after screening for possible target interactors by means of a yeast two-hybrid screen, several methods are used to validate the initial result before carrying out functional experiments. Nowadays, non-invasive fluorescence-based methods like Bioluminescence Resonance Energy Transfer (BRET) and Fluorescence Resonance Energy Transfer (FRET) are widely used in the study of protein-protein interactions in living cells. In the present review, we address the individual strengths and weaknesses of both RET approaches, providing information on their possible future use in the study of G protein-coupled receptor oligomerization.     

Effect of bovine serum albumin on the isolation of boar spermatozoa and their fertility

A procedure for isolation of subpopulations of highly motile spermatozoa from ejaculates of several mammalian species was modified for use with boar semen. An initial trial was conducted to test the effectiveness of concentrations of 3 to 15% Bovine Serum Albumin (BSA) as an isolation medium. A concentration of 15% BSA resulted in isolation of a population of boar spermatozoa with 70% motility compared with an estimate of 53.5% determined after collection. Although there was a variable effect of isolation on the rate of forward movement, sperm cell concentration decreased (P<.05) as BSA level increased.To test the fertilizing capacity of isolated boar spermatozoa, 16 sows were randomly alloted to either a treated group bred with isolated spermatozoa (12% BSA) or control group bred with raw semen. Both the percent motile spermatozoa (64.1%) and the rate of forward movement (2.9) were increased (P<.01) after isolation in BSA compared with initial values obtained at collection (51.4% and 2.3). Farrowing rates (71.4% and 75% for treated and control groups, respectively) were not significantly different, and the sex ratio of the offspring was not altered by the procedure.     

Effects of ammonium sulfate on the unfolding and refolding of the variable and constant fragments of an immunoglobulin light chain

The equilibria and kinetics of unfolding and refolding by guanidine hydrochloride of the VL and CL fragments of a type kappa immunoglobulin light chain were studied in the presence of ammonium sulfate using circular dichroism and tryptophyl fluorescence at pH 7.5 and 25 degrees C. The unfolding equilibria of the VL and CL fragments were described in terms of the two-state transition. The midpoints of unfolding in the absence of ammonium sulfate were at 0.9 and 1.2 M guanidine hydrochloride for the CL and VL fragments respectively. The transition curves were shifted to higher concentrations of guanidine hydrochloride by 1.4 and 1.6 M for the CL and VL fragments, respectively, per mole of ammonium sulfate. Unfolding reactions of the VL and CL fragments in 3 M guanidine hydrochloride followed first-order kinetics, and the rate constants for the two proteins were both greatly decreased by the presence of ammonium sulfate. The refolding reaction of the CL fragment in 0.3 M guanidine hydrochloride consisted of two phases, and the rate constants were increased a little by the presence of ammonium sulfate. The refolding reaction of the VL fragment in 0.3 M guanidine hydrochloride followed first-order kinetics, and the rate was not affected by the presence of ammonium sulfate. These results showed that ammonium sulfate stabilizes the CL and VL fragments mainly by decreasing the unfolding rate.     

The N-linked sugar chains of human immunoglobulin G: their unique pattern, and their functional roles

In contrast to other serum glycoproteins, the majority of the N-linked sugar chains of human serum IgG are not sialylated. In addition, extremely high micro-heterogeneity occurs in the serum IgG sugar chains. This micro-heterogeneity is mainly produced by the presence or absence of the two galactoses, the bisecting GlcNAc, and the fucose residue. Interesting evidence is that the molar ratio of each sugar chain of the serum IgG samples is quite constant in healthy individuals. By adding the information of the characteristic feature of the sugar patterns of myeloma IgG samples and glycosylated Bence Jones proteins, which are the products of monoclonal B-cells, it was proposed that B-cells in the human blood are a mixture of clones equipped with different sets and ratios of glycosyltransferases. It was also proposed that each glycoform of IgG might have a different function. This hypothesis was realized by the comparative studies of the function of IgG samples before and after removal of galactose residues, fucose residue, or sialic acid residues.     

The comparative effects of feeding ammonium carbonate, ammonium sulfate, and ammonium chloride on urinary calcium excretion in the rat

When either sulfate or chloride is added to the diet, the resulting acid load causes a rise in urinary calcium excretion. There is, however, the possibility that sulfate, which has been shown to complex renal tubular calcium, will further decrease renal calcium reabsorption and thus produce a greater calciuria than chloride. Because addition of a fixed cation (e.g., sodium) to the diet may also stimulate calciuresis, experiments were conducted using metabolizable ammonium to minimize cation effects. Ammonium salts of sulfate, chloride, and carbonate (control) were added to the diets of male rats at 0.3 mequiv./g weight of diet. Twenty-four hour excretion rates of calcium, sulfate, chloride, and net acid were measured at various intervals up to 1 month. As expected, the chloride and sulfate diets were both associated with significantly elevated urine calcium and net acid excretion as compared with controls. However, those fed sulfate exhibited significantly less calcium and acid excretion and absorbed a smaller proportion of the anion load than those given chloride. In a second experiment, the amounts of supplemental sulfate and chloride were adjusted so that total absorptions were similar. At 2 weeks, both calcium and acid excretions in the fixed anion groups were no longer significantly different. Thus, in chronic feeding trials, there appears to be no measurable difference in the calciuretic properties of sulfate and chloride anions.     

Production of a Pseudomonas lipase in n-alkane substrate and its isolation using an improved ammonium sulfate precipitation technique

Among the various lipidic and non-lipidic substances, normal alkanes within the chain lengths of C-12 to C-20 served as the best carbon substrates for the production of extracellular lipase by Pseudomonas species G6. Maximum lipase production of 25 U/ml of the culture broth was obtained by using n-hexadecane as the sole carbon substrate. The optimum pH of 8 and temperature of 34 + 1 degrees C were demonstrated for the production of lipase in n-hexadecane substrate. The optimum concentration of iron, which played a critical role on the lipase production, was found to be 0.25 mg/l. Lipase production could be enhanced to nearly 2.4-fold by using tributyrin at a concentration of 0.05% (v/v) in the culture medium. High recovery of the lipase protein (83%) from the culture broth was achieved by treating the culture supernatant with Silicone 21 Defoamer followed by ammonium sulfate (60% saturation) fractionation.     

Analysis and use of the serum albumin binding domains of streptococcal protein G

Streptococcal protein G is an IgG-binding receptor with a molecular weight of 63 kDa as predicted from the sequence of the corresponding gene. Here we show that a truncated recombinant protein of 23 kDa still has IgG-binding capacity and also interacts specifically with human serum albumin (HSA). This demonstrates that protein G is a bifunctional receptor. To investigate the structures needed for IgG- and albumin-binding, different parts of the receptor molecule were produced in E. coli using a coupled expression/secretion system. Affinity chromatography, using IgG or HSA immobilized on Sepharose, showed that the two binding activities are structurally separated. From these experiments, it was concluded that a region of 64 amino acid residues is sufficient for albumin-binding. The structure of this part of the protein suggests either a divalent or a trivalent binding capacity. The specific interaction to albumin was used to purify a heterologous protein by affinity chromatography to yield a pure fusion protein in a one-step procedure. The implication of this novel affinity system as a tool to facilitate protein immobilization and purification is discussed.