Glargine and degludec: Solution behaviour of higher dose synthetic insulins.

Single, double and triple doses of the synthetic insulins glargine and degludec currently used in patient therapy are characterised using macromolecular hydrodynamic techniques (dynamic light scattering and analytical ultracentrifugation) in an attempt to provide the basis for improved personalised insulin profiling in patients with diabetes. Using dynamic light scattering and sedimentation velocity in the analytical ultracentrifuge glargine was shown to be primarily dimeric under solvent conditions used in current formulations whereas degludec behaved as a dihexamer with evidence of further association of the hexamers ("multi-hexamerisation"). Further analysis by sedimentation equilibrium showed that degludec exhibited reversible interaction between mono- and-di-hexamer forms.

Distinct Contributions of Tryptophan Residues within the Dimerization Domain to Nanog Function.

The level of the transcription factor Nanog directly determines the efficiency of mouse embryonic stem cell self-renewal. Nanog protein exists as a dimer with the dimerization domain composed of a simple repeat region in which every fifth residue is a tryptophan, the tryptophan repeat (WR). Although WR is necessary to enable Nanog to confer LIF-independent self-renewal, the mechanism of dimerization and the effect of modulating dimerization strength have been unclear.

Application of novel analytical ultracentrifuge analysis to solutions of fungal mannans.

Polysaccharides, the most abundant biopolymers, are required for a host of activities in lower organisms, animals, and plants. Their solution characterization is challenging due to their complex shape, heterogeneity, and size. Here, recently developed data analysis approaches were applied for traditional sedimentation equilibrium and velocity methods in order to investigate the molar mass distribution(s) of a subtype of polysaccharide, namely, mannans from four Candida spp.

Location dependent coordination chemistry and MRI relaxivity, in designed lanthanide coiled coils.

Herein, we establish for the first time the design principles for lanthanide coordination within coiled coils, and the important consequences of binding site translation. By interrogating design requirements and by systematically translating binding site residues, one can influence coiled coil stability and more importantly, the lanthanide coordination chemistry. A 10 Å binding site translation along a coiled coil, transforms a coordinatively saturated Tb(Asp)(Asn) site into one in which three exogenous water molecules are coordinated, and in which the Asn layer is no longer essential for binding, Tb(Asp)(HO).

A multilaboratory comparison of calibration accuracy and the performance of external references in analytical ultracentrifugation.

Analytical ultracentrifugation (AUC) is a first principles based method to determine absolute sedimentation coefficients and buoyant molar masses of macromolecules and their complexes, reporting on their size and shape in free solution. The purpose of this multi-laboratory study was to establish the precision and accuracy of basic data dimensions in AUC and validate previously proposed calibration techniques. Three kits of AUC cell assemblies containing radial and temperature calibration tools and a bovine serum albumin (BSA) reference sample were shared among 67 laboratories, generating 129 comprehensive data sets.

Hydrodynamic characterisation of chitosan and its interaction with two polyanions: DNA and xanthan.

Chitosan, a soluble polycationic derivative of insoluble chitin, has been widely considered for use in the food, cosmetic and pharmaceutical industries. Commercial ("C") and in-house laboratory ("L") prepared chitosan samples extracted from crustaceous shells with different molecular weight and degrees of acetylation (25% and 15%) were compared with regards to (i) weight-average molecular weight (Mw); (ii) sedimentation coefficient (s(o)(20,w)) distribution, and (iii) intrinsic viscosity ([η]). These parameters were estimated using a combination of analytical ultracentrifugation (AUC), size exclusion chromatography coupled to multi-angle laser light scattering (SEC-MALS) and differential pressure viscometry.

A review of modern approaches to the hydrodynamic characterisation of polydisperse macromolecular systems in biotechnology.

This short review considers the range of modern techniques for the hydrodynamic characterisation of macromolecules - particularly large glycosylated systems used in the food, biopharma and healthcare industries. The range or polydispersity of molecular weights and conformations presents special challenges compared to proteins. The review is aimed, without going into any great theoretical or methodological depth, to help the Industrial Biotechnologist choose the appropriate methodology or combination of methodologies for providing the detail he/she needs for particular applications.

Protein-like fully reversible tetramerisation and super-association of an aminocellulose.

Unusual protein-like, partially reversible associative behaviour has recently been observed in solutions of the water soluble carbohydrates known as 6-deoxy-6-(ω-aminoalkyl)aminocelluloses, which produce controllable self-assembling films for enzyme immobilisation and other biotechnological applications. Now, for the first time, we have found a fully reversible self-association (tetramerisation) within this family of polysaccharides. Remarkably these carbohydrate tetramers are then seen to associate further in a regular way into supra-molecular complexes.

SEDFIT-MSTAR: molecular weight and molecular weight distribution analysis of polymers by sedimentation equilibrium in the ultracentrifuge.

Sedimentation equilibrium (analytical ultracentrifugation) is one of the most inherently suitable methods for the determination of average molecular weights and molecular weight distributions of polymers, because of its absolute basis (no conformation assumptions) and inherent fractionation ability (without the need for columns or membranes and associated assumptions over inertness). With modern instrumentation it is also possible to run up to 21 samples simultaneously in a single run. Its application has been severely hampered because of difficulties in terms of baseline determination (incorporating estimation of the concentration at the air/solution meniscus) and complexity of the analysis procedures.

MultiSig: a new high-precision approach to the analysis of complex biomolecular systems.

MultiSig is a newly developed mode of analysis of sedimentation equilibrium (SE) experiments in the analytical ultracentrifuge, having the capability of taking advantage of the remarkable precision (~0.1% of signal) of the principal optical (fringe) system employed, thus supplanting existing methods of analysis through reducing the 'noise' level of certain important parameter estimates by up to orders of magnitude. Long-known limitations of the SE method, arising from lack of knowledge of the true fringe number in fringe optics and from the use of unstable numerical algorithms such as numerical differentiation, have been transcended.

An analytical ultracentrifugation based study on the conformation of lambda carrageenan in aqueous solution.

The conformation and heterogeneity of lambda-carrageenan, a sulphonated galactan from red seaweed, solubilised in aqueous solvent with the assistance of microwave irradiation, has been assessed by a combination of analytical ultracentrifugation, size-exclusion chromatography, light scattering and capillary viscometry. Preparations appeared generally unimodal on the basis of sedimentation coefficient distributions from sedimentation velocity although at the highest concentrations a shoulder appears with a sedimentation coefficient approximately 1.1 times greater than that of the main component.

Molecular weight distribution analysis by ultracentrifugation: adaptation of a new approach for mucins.

Mucins are the key macromolecular component of mucus, nature's natural lubricant, and one of the most important physical properties is their molecular weight distribution. A new approach for polydisperse polymers was recently published based on sedimentation velocity in the analytical ultracentrifuge and converts a distribution of sedimentation coefficient g(s) vs. s plot into a distribution of molecular weight utilising the power-law or scaling relationship between the sedimentation coefficient and molecular weight, s=κsMw(b) where s is the sedimentation coefficient, Mw is the weight average molecular weight and κs and b are characteristic coefficients related to conformation.

An asymmetric and slightly dimerized structure for the tetanus toxoid protein used in glycoconjugate vaccines.

Tetanus toxoid protein has been characterized with regard oligomeric state and hydrodynamic (low-resolution) shape, important parameters with regard its use in glycoconjugate vaccines. From sedimentation velocity and sedimentation equilibrium analysis in the analytical ultracentrifuge tetanus toxoid protein is shown to be mostly monomeric in solution (~86%) with approximately 14% dimer. The relative proportions do not appear to change significantly with concentration, suggesting the two components are not in reversible equilibrium.

T-shaped arrangement of the recombinant agrin G3-IgG Fc protein.

Agrin is a large heparin sulphate proteoglycan with multiple domains, which is located in the extracellular matrix. The C-terminal G3 domain of agrin is functionally one of the most important domains. It harbors an α-dystroglycan binding site and carries out acetylcholine receptor clustering activities.

Ultra-weak reversible protein-protein interactions.

Ultra-weak interactions (K(d)>100μM) between proteins have in the last decade become an increasing focus of attention in cell biology, especially in relation to cell-cell interactions and signalling processes. Methods for their quantitative definition are reviewed. NMR spectroscopy plays a major role in this area, as it not only can define interactions as weak or weaker than 3mM, but in favourable cases structural information concerning the complex can be yielded.

Insight into protein-protein interactions from analytical ultracentrifugation.

Analytical ultracentrifugation is a free solution technique with no supplementary immobilization, columns or membranes required, and can be used to study self-association and hetero-interactions, stoichiometry, reversibility and interaction strength across a very large dynamic range (dissociation constants from 10(-12) M to 10(-1) M). In the present paper, we review some of the advances that have been made in the two different types of sedimentation experiment--sedimentation equilibrium and sedimentation velocity--for the analysis of protein-protein interactions and indicate how major complications such as thermodynamic and hydrodynamic non-ideality can be dealt with.

Evaluation of the information content of sedimentation equilibrium data in self-interacting systems.

Fitting r = f(c) as opposed to the usual c = f(r) to the inverted form of the sedimentation equilibrium equation for interacting solute (INVEQ algorithm), it is shown by detailed simulation and by experimentation that stable, simultaneous estimates can be retrieved for both virial (2nd BM/3rd CM) and specific interaction (K(a)) terms. In suitable systems estimates for two distinct second virial (BM) and single K(a) terms can equally be defined. Whilst cell loading level is critical, noise level in the interference fringe data is shown to have surprisingly little influence on these outcomes.

The central portion of factor H (modules 10-15) is compact and contains a structurally deviant CCP module.

The first eight and the last two of 20 complement control protein (CCP) modules within complement factor H (fH) encompass binding sites for C3b and polyanionic carbohydrates. These binding sites cooperate self-surface selectively to prevent C3b amplification, thus minimising complement-mediated damage to host. Intervening fH CCPs, apparently devoid of such recognition sites, are proposed to play a structural role.

Structure and heterogeneity of gliadin: a hydrodynamic evaluation.

A study of the heterogeneity and conformation in solution [in 70% (v/v) aq. ethanol] of gliadin proteins from wheat was undertaken based upon sedimentation velocity in the analytical ultracentrifuge, analysis of the distribution coefficients and ellipsoidal axial ratios assuming quasi-rigid particles, allowing for a range of plausible time-averaged hydration values. All classical fractions (alpha, gamma, omega(slow), omega(fast)) show three clearly resolved components.

Oligomerisation of pneumolysin on cholesterol crystals: similarities to the behaviour of polyene antibiotics.

Pneumolysin is a cytolytic toxin of Streptococcus pneumoniae, a causative agent of pneumonia and meningitis. The prepore and pore states of pneumolysin have recently been investigated by cryo-electron microscopy and atomic force microscopy, confirming the existence of arc-shaped as well as ring-form oligomers. Here we provide further insights into the pneumolysin oligomer by studying the interaction of pneumolysin with cholesterol crystals, comparing the results to those obtained for polyene antibiotics, which also bind cholesterol.

The pluripotency rheostat Nanog functions as a dimer.

The defining activity of the homeodomain protein Nanog is the ability to confer cytokine-independent self-renewal upon ES (embryonic stem) cells in which it is overexpressed. However, the biochemical basis by which Nanog achieves this function remains unknown. In the present study, we show that Nanog dimerizes through a functionally critical domain.

TPR-Mediated self-association of plant SGT1.

The tetratricopeptide repeat (TPR) domain mediates inter-protein associations in a number of systems. The domain is also thought to mediate oligomerization of some proteins, but this has remained controversial, with conflicting data appearing in the literature. By way of investigating such TPR-mediated self-associations we used a variety of biophysical techniques to characterize purified recombinant Sgt1, a TPR-containing protein found in all eukaryotes that is involved in a broad range of biological processes, including kinetochore assembly in humans and yeast and disease resistance in plants.

Effect of PEGylation on the solution conformation of antibody fragments.

Covalent attachment of poly(ethylene glycol) (PEG) to therapeutic antibody fragments has been found effective in prolonging the half-life of the protein molecule in vivo. In this study analytical ultracentrifugation (AUC) in combination with small angle X-ray scattering (SAXS) has been applied to a number of antibody fragments and to their respective PEGylated conjugates. Despite the large increase in molecular weight due to the attachment of a 20-40 kDa PEG moiety, the PEGylated conjugates have smaller sedimentation coefficients, s, than their parent antibody fragments, due to a significant increase in frictional ratio f/f(o) (from approximately 1.

The effect of a point mutation on the stability of IgG4 as monitored by analytical ultracentrifugation.

There is presently considerable interest in the state of aggregation and biophysical integrity of antibody preparations, and recent advances in the analysis of data from the analytical ultracentrifuge renders it a powerful probe of these stability phenomena, under both storage and freeze-thaw conditions. Solutions of a wild-type IgG4 antibody and a single amino acid hinge mutant IgG4m (serine residue 241 converted to proline) were exposed to different accelerated stress conditions, namely (i) elevated temperature storage for various periods (up to 59 days at 37 degrees C) or (ii) a series of freeze-thaw cycles (storage at -80 degrees C then incubation at 20 degrees C for 1 h under different conditions). Analysis using the nondisruptive probe of sedimentation velocity in the analytical ultracentrifuge indicated that for both antibodies the monomer was always the most common species present whatever storage regime had been used.