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. Unlike glargine, degludec showed strong thermodynamic non-ideality, but this was suppressed by the addition of salt. With such large injectable doses of synthetic insulins remaining in the physiological system for extended periods of time, in some case 24-40 hours, double and triple dose insulins may impact adversely on personalised insulin profiling in patients with diabetes.

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. The molecular weight distributions of these mannans were studied using two recently developed equilibrium approaches: SEDFIT-MSTAR and MULTISIG, resulting in corroboratory distribution profiles. Additionally, sedimentation velocity data for all four mannans, analyzed using ls-g*(s) and Extended Fujita approaches, suggest that two of the fungal mannans (FM-1 and FM-3) have a unimodal distribution of molecular species whereas two others (FM-2 and FM-4) displayed bi-modal and broad distributions, respectively: this demonstrates considerable molecular heterogeneity in these polysaccharides, consistent with previous observations of mannans and polysaccharides in general. These methods not only have applications for the characterization of mannans but for other biopolymers such as polysaccharides, DNA, and proteins (including intrinsically disordered proteins).

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. Here we couple mutagenesis with functional and dimerization assays to show that the number of tryptophans within the WR is linked to the strength of homodimerization, Sox2 heterodimerization and self-renewal activity. A reduction in the number of tryptophan residues leads initially to a gradual reduction in activity before a precipitous reduction in activity occurs upon reduction in tryptophan number below eight. Further functional attrition follows subsequent tryptophan number reduction with substitution of all tryptophan residues ablating dimerization and self-renewal function completely. A strong positional influence of tryptophans exists, with residues at the WR termini contributing more to Nanog function, particularly at the N-terminal end. Limited proteolysis demonstrates that a structural core of Nanog encompassing the homeodomain and the tryptophan repeat can support LIF-independent colony formation. These results increase understanding of the molecular interactions occurring between transcription factor subunits at the core of the pluripotency gene regulatory network and will enhance our ability to control pluripotent cell self-renewal and differentiation.

Location dependent coordination chemistry and MRI relaxivity, in de novo 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)3(Asn)3 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)3(H2O)3. This has a profound impact on the relaxivity of the analogous Gd(iii) coiled coil, with more than a four-fold increase in the transverse relaxivity (21 to 89 mM-1 s-1), by bringing into play, in addition to the outer sphere mechanism present for all Gd(iii) coiled coils, an inner sphere mechanism. Not only do these findings warrant further investigation for possible exploitation as MRI contrast agents, but understanding the impact of binding site translation on coordination chemistry has important repercussions for metal binding site design, taking us an important step closer to the predictable and truly de novo design of metal binding sites, for new functional applications.

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. Polydisperse distributions were seen from sedimentation coefficient distributions and elution profiles from SEC-MALS. Mw values obtained for each sample by sedimentation equilibrium measurements were in excellent agreement with those obtained from SEC-MALS. Mark-Houwink-Kuhn-Sakurada (MHKS) and Wales van Holde analyses of the data all suggest a semi-flexible conformation. The principle of co-sedimentation was then used to monitor the interactions of the two different molecular weights of L chitosans with two polyanions, DNA and xanthan (another double helical high molecular weight molecule). Interactions were clearly observed and then quantified from the changes in the sedimentation coefficient distribution of the mixture compared to unmixed controls using sedimentation velocity. The interactions appeared to show a strong dependence on molecular weight. The relevance of this for DNA condensation applications is indicated.

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. Fully reversible oligomerisation has been hitherto completely unknown for carbohydrates and instead resembles in some respects the assembly of polypeptides and proteins like haemoglobin and its sickle cell mutation. Our traditional perceptions as to what might be considered "protein-like" and what might be considered as "carbohydrate-like" behaviour may need to be rendered more flexible, at least as far as interaction phenomena are concerned.

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.

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. We describe a new method for baseline determination based on a smart-smoothing principle and built into the highly popular platform SEDFIT for the analysis of the sedimentation behavior of natural and synthetic polymer materials. The SEDFIT-MSTAR procedure - which takes only a few minutes to perform - is tested with four synthetic data sets (including a significantly non-ideal system), a naturally occurring protein (human IgG1) and two naturally occurring carbohydrate polymers (pullulan and λ-carrageenan) in terms of (i) weight average molecular weight for the whole distribution of species in the sample (ii) the variation in "point" average molecular weight with local concentration in the ultracentrifuge cell and (iii) molecular weight distribution.

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 approach to data analysis, akin to 'spatial filtering', has been developed, and shown by both simulation and practical application to be a powerful aid to the precision with which near-monodisperse systems can be analysed, potentially yielding information on protein-solvent interaction. For oligo- and poly-disperse systems the information returned includes precise average mass distributions over both cell radial and concentration ranges and mass-frequency histograms at fixed radial positions. The application of MultiSig analysis to various complex heterogenous systems and potentially multiply-interacting carbohydrate oligomers is described.

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. Even under conditions commensurate with charge suppression simple linear regression was insufficient to represent non-ideal concentration dependence and the extraction of the Grálen concentration dependence parameter ks. A more general fitting algorithm was therefore employed. Mark-Houwink-Kuhn-Sakurada analysis of the change in intrinsic viscosity [η] with molecular weight, together with the Wales-van Holde ratio (combination of ks with [η]) point to an extended flexible conformation for lambda-carrageenan in the (weight average) molecular weight range Mw=340,000-870,000g/mol. The origin of the larger sedimentation coefficient component appearing at the higher concentrations is considered.

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. We investigate the possibility of using a large database of previously published values of s an M to define κs and b for both aqueous solution and aqueous solution supplemented by 6M guanidine hydrochloride (a solvent which helps to minimise sample degradation). These values are then applied to a study of the molecular weight distributions of preparations of human gastric mucin in the different solvents and at different stages of purification.

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. Hydrodynamic solution conformation studies based on high precision viscometry, combined with sedimentation data show the protein to be slightly extended conformation in solution with an aspect ratio ~3. The asymmetric structure presents a greater surface area for conjugation with polysaccharide than a more globular structure, underpinning its popular choice as a conjugation protein for glycoconjugate vaccines.

Lack of evidence from studies of soluble protein fragments that Knops blood group polymorphisms in complement receptor-type 1 are driven by malaria.

Complement receptor-type 1 (CR1, CD35) is the immune-adherence receptor, a complement regulator, and an erythroid receptor for Plasmodium falciparum during merozoite invasion and subsequent rosette formation involving parasitized and non-infected erythrocytes. The non-uniform geographical distribution of Knops blood group CR1 alleles Sl1/2 and McC(a/b) may result from selective pressures exerted by differential exposure to infectious hazards. Here, four variant short recombinant versions of CR1 were produced and analyzed, focusing on complement control protein modules (CCPs) 15-25 of its ectodomain. These eleven modules encompass a region (CCPs 15-17) key to rosetting, opsonin recognition and complement regulation, as well as the Knops blood group polymorphisms in CCPs 24-25. All four CR1 15-25 variants were monomeric and had similar axial ratios. Modules 21 and 22, despite their double-length inter-modular linker, did not lie side-by-side so as to stabilize a bent-back architecture that would facilitate cooperation between key functional modules and Knops blood group antigens. Indeed, the four CR1 15-25 variants had virtually indistinguishable affinities for immobilized complement fragments C3b (K(D) = 0.8-1.1 µM) and C4b (K(D) = 5.0-5.3 µM). They were all equally good co-factors for factor I-catalysed cleavage of C3b and C4b, and they bound equally within a narrow affinity range, to immobilized C1q. No differences between the variants were observed in assays for inhibition of erythrocyte invasion by P. falciparum or for rosette disruption. Neither differences in complement-regulatory functionality, nor interactions with P. falciparum proteins tested here, appear to have driven the non-uniform geographic distribution of these alleles.

Protein engineered variants of hepatocyte growth factor/scatter factor promote proliferation of primary human hepatocytes and in rodent liver.

BACKGROUND & AIMS: Hepatocyte growth factor/scatter factor (HGF/SF) stimulates hepatocyte DNA synthesis and protects against apoptosis; in vivo it promotes liver regeneration and reduces fibrosis. However, its therapeutic value is limited by its complex domain structure, high cost of production, instability, and poor tissue penetration due to sequestration by heparin sulfate proteoglycans (HSPGs). METHODS: Using protein engineering techniques, we created a full-length form of HGF/SF (called HP21) and a form of the small, naturally occurring HGF/SF fragment, NK1 (called 1K1), which have reduced affinity for HSPG. We characterized the stability and proliferative and anti-apoptotic effects of these variants in primary human hepatocytes and in rodents. RESULTS: Analytical ultracentrifugation showed that 1K1 and NK1 were more stable than the native, full-length protein. All 4 forms of HGF/SF induced similar levels of DNA synthesis in human hepatocytes; 1K1 and NK1 required heparin, an HSPG analogue, for full agonistic activity. All the proteins reduced levels of Fas ligand-mediated apoptosis, reducing the activity of caspase-3/7 and cleavage of poly(adenosine diphosphate-ribose) polymerase. 1K1 was more active than NK1 in rodents; in healthy mice, 1K1 significantly increased hepatocyte DNA synthesis, and in mice receiving carbon tetrachloride, it reduced fibrosis. In rats, after 70% partial hepatectomy, daily administration of 1K1 for 5 days significantly increased liver mass and the bromodeoxyuridine labeling index compared with mice given NK1. CONCLUSIONS: 1K1, an engineered form of the small, naturally occurring HGF/SF fragment NK1, has reduced affinity for HSPG and exerts proliferative and antiapoptotic effects in cultured hepatocytes. In rodents, 1K1 has antifibrotic effects and promotes liver regeneration. The protein has better stability and is easier to produce than HGF/SF and might be developed as a therapeutic for acute and chronic liver disease.

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. In the present study, we have fused the G3 domain of agrin to an IgG Fc domain to produce a G3-Fc fusion protein that we intend to use as a tool to investigate new binding partners of agrin. As a first step of the study, we have characterized the recombinant fusion protein using a multidisciplinary approach using dynamic light scattering, analytical ultracentrifugation and small angle X-ray scattering (SAXS). Interestingly, our SAXS analysis using the high-resolution structures of G3 and Fc domain as models indicates that the G3-Fc protein forms a T-shaped molecule with the G3 domains extruding perpendicularly from the Fc scaffold. To validate our models, we have used the program HYDROPRO to calculate the hydrodynamic properties of the solution models. The calculated values are in excellent agreement with those determined experimentally.

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. Free solution technologies mostly fail when addressed to such systems. The AUC has the highest practical capability, but evaluation of the data to yield K(a) values is complicated by the presence of thermodynamic/hydrodynamic effects of a comparable order of magnitude. These effects can however be computationally removed by means of suitable algorithms, and K(d) values of up to 50mM can be characterised. The relative merits of velocity and equilibrium approaches are discussed, and both are shown to have particular advantages.

Bioequivalence comparison of a new freezing bag (CryoMACS(®)) with the Cryocyte(®) freezing bag for cryogenic storage of human hematopoietic progenitor cells.

BACKGROUND AIMS: We investigated two different plastic freezing bags, namely the most recently U.S. Food and Drug Administration (FDA)-approved CryoMACS(®) freezing bag (200-074-402) from Miltenyi Biotec and the familiar Cryocyte(®) freezing bag (R4R9955) from (Baxter Healthcare, Deerfield, IL, United States) for the cryogenic storage of human hematopoietic progenitor cells (HPC). METHODS: The study material consisted of 12 frozen HPC pairs (= 24 transplant units) that were no longer needed for autologous treatment of patients. After thawing, one unit of a pair was transferred into the Miltenyi (M) bag; the other unit remained in the original Baxter (B) bag. After refreezing both units, all units were stored again under cryogenic conditions either partially immersed in liquid nitrogen (n = 22) or in the vapor phase over liquid nitrogen, n = 2, <-170°) before thawing. RESULTS: The correlation coefficients (r) between the results obtained from the two bag types were high for white blood cells (WBC) content (r = 0.98), mononuclear cells (MNC) (r = 0.97), lymphocytes (r = 0.98), monocytes (r = 0.96), membrane integrity (r = 0.93), concentration of 'free' hemoglobin (r = 0.97) and hemolysis rate (r = 0.95). With regard to clonogenicity, there were no significant differences (Student's paired t-test) for the three parameters investigated [i.e. total number of colonies, including the numbers of burst-forming units-erythroid (BFU-E) and colony-forming units-granulocyte-macrophage (CFU-GM) colonies, respectively). CONCLUSIONS: The CryoMACS freezing bag 200-074-402 is bioequivalent to the Cryocyte freezing container R4R9955. An advantageous feature of the CryoMACS is that its double-sterile wrapping provides additional safety regarding potential cross-contamination during cryogenic storage.

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.