Hydrodynamic modelling of protein conformation in solution: ELLIPS and HYDRO

The last three decades has seen some important advances in our ability to represent the conformation of proteins in solution on the basis of hydrodynamic measurements. Advances in theoretical modeling capabilities have been matched by commensurate advances in the precision of hydrodynamic measurements. We consider the advances in whole-body (simple ellipsoid-based) modeling—still useful for providing an overall idea of molecular shape, particularly for those systems where only a limited amount of data is available—and outline the ELLIPS suite of algorithms which facilitates the use of this approach. We then focus on bead modeling strategies, particularly the surface or shell–bead approaches and the HYDRO suite of algorithms. We demonstrate how these are providing great insights into complex issues such as the conformation of immunoglobulins and other multi-domain complexes.     

Bead modeling using HYDRO and SOLPRO of the conformation of multisubunit proteins: sunflower and rape-seed 11S globulins

Oil seed globulins from sunflower and rape seed are multi-subunit, oligomeric proteins whose native 11S form is a hexamer. In this work we try to determine the spatial structure in which the six subunits of 11S globulin are arranged. Experimental values of solution properties, including radius of gyration, sedimentation and diffusion coefficients and intrinsic viscosity, are compared with theoretical predictions for hexamers of various geometries. Bead model calculations of solution properties are carried out using the HYDRO and SOLPRO computer programs. A most compact shape, the regular octahedron, is the hexameric structure that fits best the experimental values.     

Macromolecular conformation of chitosan in dilute solution: A new global hydrodynamic approach

Chitosans of different molar masses were prepared by storing freshly prepared samples for up to 6 months at either 4, 25 or 40 °C. The weight-average molar masses, M_w and intrinsic viscosities, [η] were then measured using size exclusion chromatography coupled to multi-angle laser light scattering (SEC-MALLS) and a “rolling ball” viscometer, respectively.The solution conformation of chitosan was then estimated from:

(a) the Mark–Houwink–Kuhn–Sakurada (MHKS) power law relationship [η] = kM_w^a and

(b) the persistence length, L_p calculated from a new approach based on equivalent radii [Ortega, A., & Garcia de la Torre, J. (2007). Equivalent radii and ratios of radii from solution properties as indicators of macromolecular conformation, shape, and flexibility. Biomacromolecules, 8, 2464–2475].

Both the MHKS power law exponent (a = 0.95 ± 0.01) and the persistence length (L_p = 16 ± 2 nm) are consistent with a semi-flexible rod type (or stiff coil) conformation for all 33 chitosans studied. A semi-flexible rod conformation was further supported by the Wales–van Holde ratio, the translational frictional ratio and sedimentation conformation zoning.     

The compact domain conformation of human Glu-plasminogen in solution.

A complete understanding of the accelerating mechanisms of plasminogen activation and fibrinolysis necessarily requires structural information on the conformational forms of plasminogen. Given the absence of high-resolution structural data on plasminogen the use of lower resolution approaches has been adopted. Two such approaches have previously indicated a compact conformation of Glu-plasminogen (Tranqui, L., Prandini, M., and Chapel, A. (1979) Biol. Cellulaire, 34, 39-42; Bányai, L. and Patthy, L. (1985) Biochim. Biophys. Acta, 832, 224-227) whereas a third has suggested a fairly extended conformation (Mangel, W., Lin, B. and Ramakrishnan, V. (1990) Science, 248, 69-73). Native Glu-plasminogen has been investigated using small-angle X-ray scattering (SAXS) experiments. It is concluded that this molecule in solution is compact (radius of gyration, RG 3.05 +/- 0.02 nm and maximum intramolecular distance, Im 9.1 +/- 0.3 nm) and that the data are consistent with the right-handed spiral structure observed using electron microscopy by Tranqui et al. (1979). A spiral structure of native plasminogen would have important implications for the conformational response of plasminogen to fibrin and concomitant stimulation of plasminogen activation.     

Post-translational regulation of expression and conformation of an immunoglobulin domain in yeast surface display

Display of heterologous proteins on the surface of Saccharomyces cerevisiae is increasingly being exploited for directed evolution because of straightforward cell screens. However, yeast post-translationally modifies proteins in ways that must be factored into library engineering and refinement. Here, we express the extracellular immunoglobulin domain of an ubiquitous mammalian membrane protein, CD47, which is implicated in cancer, immunocompatibility, and motility. CD47 has multiple sites of glycosylation and a core disulfide bond. We assess the effects of both of these post-translational modifications on expression and antibody binding. CD47's extracellular domain is fused to the yeast mating protein Aga2p on the cell wall, and the resulting fusion protein binds several key antibodies, including a conformation-sensitive antibody. Site-by-site mutagenesis of CD47's five N-linked glycosylation sites progressively decreases expression levels on yeast, but folding appears stable. Cysteine mutations disrupt the expected core disulfide, and also decrease protein expression levels, though not to the extent seen with complete deglycosylation. However, with the core disulfide mutants, antibody binding proves to be lower than expected from expression levels and glycosylation is clearly reduced compared to wild-type. The results indicate that glycosylation regulates heterologous display on yeast more than core disulfides do and thus suggest bounds on directed evolution by post-translational processing.     

Correlation between the antitumor activity of a polysaccharide schizophyllan and its triple-helical conformation in dilute aqueous solution

Eight samples of a polysaccharide schizophyllan ranging in weight-average molecular weight Mw (in water) from 5 x 10(3) to 1.3 x 10(5) were prepared and their antitumor activity (expressed in terms of the tumor inhibition ratio) against Sarcoma 180 ascites, intrinsic viscosities [eta], and gel-filtration chromatograms in aqueous solution were determined. The tumor inhibition ratio was essentially unity for samples with Mw higher than 9 x 10(4), but reduced to zero or even to a negative value when Mw was lower than 10(4). The [eta] data combined with the chromatographic data showed that above Mw approximately 9 x 10(4) the predominant species of schizophyllan in aqueous solution is the previously found rigid triple helix, whereas below Mw approximately 9 x 10(4) both triple helices and single chains coexist in the solution and the fraction of triple helices decreases monotonically to zero as Mw is decreased to 5 x 10(3). From these findings it was concluded that the antitumor potency of schizophyllan in water is related to the amount of triple helices relative to that of single chains.     

The effect of solvent environment on the conformation and stability of human polyclonal IgG in solution.

Stability of therapeutic IgG preparations is an important issue as adequate efficacy and safety has to be ensured throughout a long shelf life. To this end, denaturation and aggregation have to be avoided. In many cases sugars are applied for stabilizing IgG in relatively high concentration (5-10%). However, certain sugars (sucrose, maltose) are responsible for adverse effects including renal failure. In this work we reassessed the effect of pH and stabilizers to optimize the solvent environment and minimize the amount of additives without endangering quality and stability. Since both biological function and aggregation depend on the conformational properties of individual IgG molecules, two sensitive and rapid physical methods were introduced to assess conformational changes and structural stability as a function of pH and addition of standard stabilizers. It was observed that the conformational stability decreases with decreasing pH, while the resistance against aggregation improves. The optimum pH range for storage is 5.0-6.0, as a compromise between conformational stability and the tendency for oligomerization. Intriguingly, additives in physiologically acceptable concentration have no effect on the thermal stability of IgG. On the other hand, glucose or sorbitol, even at a concentration as low as 1%, have significant effect on the tertiary structure as revealed by near-UV-CD spectroscopy, reflecting changes in the environment of aromatic side-chains. Although, 0.3% leucine does not increase conformational stability, it decreases the aggregation tendency even more efficiently than 1% glucose or sorbitol. Both pH and storage temperature are decisive factors for the long-term stability of IgG solutions. An increase in the dimer content was observed upon storage at 5 degrees C which was partly reverted upon incubation at 37 degrees C. Storage at temperatures higher than 5 degrees C may help to maintain an optimal proportion of dimers. Regarding the known side effects, and their limited stabilizing capacity at low concentration, it is advisable to omit sugars at intravenous immunoglobulin (IVIG) formulation. Hydrophobic amino acids give promising alternatives.     

The study of solution conformation of allatostatins by 2-D NMR and molecular modeling

More than 70 allatostatins have been isolated from various insects and there is interest in the determination of their active conformation. We have synthesized Dippu-AST 1 (originally isolated from the cockroach Blattella germanica) and studied its conformation in solution by 2-D NMR and molecular modeling. Dippu-AST 1 belongs to the cockroach-type ASTs that have Y/FXFGL-NH(2) as the common C-terminal sequence. We found that Dippu-AST 1 forms a type I' beta-turn conformation in DMSO. We also studied the conformations of Dippu-AST 1 and six cockroach-type allatostatins in water using the molecular dynamics method. When the X amino acid in the consensus sequence Y/FXFGL-NH(2) is Ala or Ser, the allatostatin can form a typical type II beta-turn. If the X is Asp or Asn whose side chain contains a carbonyl, the allatostatin can form a type I, I' or IV beta-turn conformation; if the X is Gly, a closer gamma-turn is adopted. Our study indicates that the turn conformation is ubiquitous in cockroach-type allatostatins.     

Improved procedure for the conjugation of rabbit IgG and Fab' antibodies with beta-D-galactosidase from Escherichia coli using N,N'-o-phenylenedimaleimide.

The procedures for the conjugation of rabbit IgG and Fab' antibodies with beta-D-galactosidase from Escherichia coli using N,N'-o-phenylenedimaleimide were improved in several respects as compared with the previous methods (Eur. J. Biochem. 62, 285--292, 1976; J. Immunol. 116, 1554--1560, 1976). Maleimide residues were efficiently introduced into antibodies under an atmosphere of nitrogen; the average number of maleimide residues introduced into IgG and Fab' antibodies were 0.78 (0.65--0.86) and 0.86 (0.80--0.95) per molecule, respectively. The conjugation with the enzyme was performed at 4 degrees C at pH 6.5 for 15 or more hours. The conjugates were almost completely separated from unreacted IgG and Fab' by gel filtration. When the recoveries of IgG, Fab', and beta-D-galactosidase in the conjugates were 23-29, 35-44, and 99%, respectively, the average numbers of IgG and Fab' molecules conjugated with the enzyme were 1.5-1.7 and 2.1-2.8 per molecule, respectively. There was no significant impairment of beta-D-galactosidase activity or the activity of anti-human IgG antibody to bind to human IgG upon conjugation. However, the conjugate preparation was heterogeneous, and one-third of each preparation consisted of aggregated conjugates less useful in sandwich enzymoimmunoassay than the remaining material. The conjugate with Fab' antibody gave lower control values in sandwich enzymoimmunoassay with silicone rubber as a solid phase than that with IgG antibody.     

Assessment of conformation changes of immunoglobulin G molecules using a fluorescent probe

A proximate method for conformational changes detection in immunoglobulin G preparations used in research and medical practice is introduced. This method is based on the measurement of IgG-connected 1,8-anilinenaphtalenesulphonate fluorescent probe (ANS) time of damping. When the protein structure is broken fluorescence damping time of IgG-connected ANS rises up.     

Separation of l-lysine from dilute aqueous solution using molecular imprinting technique

In the present study, separation of l-lysine from dilute aqueous solution by solid-phase extraction based on molecular imprinting technique using a polar porogen was investigated. l-Lysine imprinted polymer (LLIP) was prepared by free radical solution polymerization of methacrylic acid and ethylene glycol dimethacrylate as functional and cross-linking monomers, in the presence of l-lysine as an imprint molecule, mixture of water and methanol as solvent and AIBN as an initiator. Non-imprinted polymer (NIP) as control was also prepared by the same procedure in the absence of template molecules. LLIP particles were applied to determine the optimum operational condition for l-lysine separation from dilute aqueous solution. In adsorption step, optimum pH and retention time were 7.8 and 90 min, while corresponding values in extraction step were 12 and 50 min, respectively. l and d-Lysine recovery by LLIP at optimum condition were found to be 96 and 58% with corresponding distribution coefficients of 8000 and 460, respectively. The retention capacity of LLIP was 27.26 mg l-lys/g of polymer at optimum condition.     

Solution conformation of wild-type and mutant IgG3 and IgG4 immunoglobulins using crystallohydrodynamics: possible implications for complement activation

We have employed the recently described crystallohydrodynamic approach to compare the time-averaged domain orientation of human chimeric IgG3wt (wild-type) and IgG4wt as well as two hinge mutants of IgG3 and an IgG4S331P (mutation from serine to proline at position 331, EU numbering) mutant of IgG4. The approach involves combination of the known shape of the Fab and Fc regions from crystallography with hydrodynamic data for the Fab and Fc fragments and hydrodynamic and small angle x-ray scattering data for the intact IgG structures. In this way, ad hoc assumptions over hydration can be avoided and model degeneracy (uniqueness problems) can be minimized. The best fit model for the solution structure of IgG3wt demonstrated that the Fab regions are directed away from the plane of the Fc region and with a long extended hinge region in between. The best fit model of the IgG3m15 mutant with a short hinge (and enhanced complement activation activity) showed a more open, but asymmetric structure. The IgG3HM5 mutant devoid of a hinge region (and also devoid of complement-activation activity) could not be distinguished at the low-resolution level from the structure of the enhanced complement-activating mutant IgG3m15. The lack of inter-heavy-chain disulphide bond rather than a significantly different domain orientation may be the reason for the lack of complement-activating activity of the IgG3HM5 mutant. With IgG4, there are significant and interesting conformational differences between the wild-type IgG4, which shows a symmetric structure, and the IgG4S331P mutant, which shows a highly asymmetric structure. This structural difference may explain the ability of the IgG4S331P mutant to activate complement in stark contrast to the wild-type IgG4 molecule which is devoid of this activity.     

The disordered conformation of kappa-carrageenan in solution as determined by NMR experiments and molecular modeling

The conformation of kappa-carrageenan in solution was studied combining 1H and 13C NMR with molecular mechanics. The experimental conditions were chosen to characterize the disordered conformation of the polymer. Particular attention has been given to explore a wide range of experimental conditions as to the dependence on solvent (water and Me2SO), polymer concentration, temperature, pH, presence of a denaturing agent (guanidinium chloride), and of ions otherwise able to induce conformational order of the carrageenan chains, either in solution (I-) or in the gel state (Rb+). Two-dimensional NOE experiments were analyzed to obtain information on internuclear distances, and molecular mechanics provided the range of energetically accessible conformations. Two inter-residue topological constraints were clearly identified: their combination is rather restricting for the chain and suggests that the disordered conformation of kappa-carrageenan is characterized by an intrinsic stiffness with high values of persistent length and characteristic ratio. They also rule out any postulated interchain hydrogen bonds. In contrast, experiments on the temperature dependence of the chemical shift in Me2SO reveal the existence of two inter-residue intramolecular H-bonds which might contribute positively to the rigidity of the polymer chain. The overall picture emerging from the present results is that of a locally elongated 'loose single helix'.     

Desalting and concentration of proteins in dilute solution using reversed-phase high-performance liquid chromatography

A rapid method for desalting and concentrating dilute protein solutions using short reversed-phase columns (3-4 cm) has been described. The recovery of proteins is usually 90-100%. The method is simple and rapid and allows the desalting and concentration of protein samples simultaneously. A wide variety of proteins in the range up to 80 kDa can be desalted in microgram to milligram amounts, and volumes up to 1 liter can be concentrated to a few milliliters by a single injection.     

Temperature and pH dependence of immunoglobulin G conformation

Previous indirect observations have indicated that IgG may change its conformation at low or high pH and at a temperature of about 35 degrees C. By means of small angle neutron scattering a change in the value of the gyration radius of two different native IgG's was observed above 44 degrees C. No similar change was detected when the sample was previously dissolved in an acidic buffer. The acidic pretreatment caused a significant decrease in the gyration radius (Rg) value measured at 20 degrees C which was partially recovered by increasing the temperature. These observations led to the assumption that the main conformational change observed appears either in the hinge region of the molecular or in the interdomain areas separating the constant and the variable domains of the Fab parts.     

Purification of animal immunoglobulin G (IgG) using peptoid affinity ligands

The availability of highly pure animal antibodies is critical in the production of diagnostic tools and biosensors. The peptoid PL16, previously isolated from an ensemble of peptoid variants of the IgG-binding peptide HWRGWV, was utilized in this work as affinity ligand on WorkBeads resin for the purification of immunoglobulin G (IgG) from a variety of mammalian sources and chicken immunoglobulin Y (IgY). The chromatographic protocol initially optimized for murine serum and ascites was subsequently employed for processing rabbit, goat and sheep, donkey, llama, and chicken sera. The PL16-WorkBeads resin proved able to recover all antibody targets with values of yield between 50 and 90%, and purity consistently above 90%. Notably, PL16 not only binds a broader spectrum of animal immunoglobulins than the reference ligands Protein A and G, but it also binds equally well with all their subclasses. Unlike the protein ligands, in fact, PL16 afforded excellent values of yield and purity of mammalian polyclonal IgG, namely murine (47 and 94%), rabbit (66.5 and 91.7%), caprine IgG (63 and 91–95%), donkey, and llama (93 and 97%), as well as chicken IgY (42 and 92%). Of notice, it is also the ability of PL16 to target monomeric IgG without binding aggregated IgG; when challenged with a mixture of monomeric and aggregated murine IgG, PL16 eluted <3% of fed aggregates, against 11–13% eluted by Protein A and G. Collectively, these results prove the potential of the proposed peptoid ligand for large-scale purification of animal immunoglobulins.