Hydrodynamic characterisation of high and low molecular weight glycans

Glycans are a diverse group of biological macromolecules that are made up of polysaccharides and glycoconjugates, such as glycoproteins and glycopeptides. The different types of glycans results in a multitude of structures, properties and functions. This investigation looks at the different hydrodynamic properties of several glycans including viscosity, sedimentation coefficient and molecular weight. These parameters are determined using techniques such as viscometry and Analytical Ultracentrifugation (AUC). A foundation on the principles of the techniques involved in hydrodynamic characterisation was provided through the use of ovalbumin, a glycoprotein that has been extensively studied. The study gave a basic understanding in Analytical Ultracentrifugation. Using the knowledge obtained from that investigation a study into possible reasons why the ‘last-resort’ glycopeptide antibiotic, vancomycin, is not commonly administered orally due to poor absorption within the gut. The study examined at the interactions of vancomycin with common macromolecules found with the gastro-intestinal tract, such as mucin, and trying to explain reasons how these interactions could inhibit the absorption of vancomycin. Finally investigations into two different species derived β-glucans, that have been shown to have medically benefiting properties, were characterised. The used of different hydrodynamic techniques yielded results that generally supported the accepted hydrodynamic properties of β-glucans, however there were results that challenged these understandings as well

Hydrodynamic characterisation of high and low molecular weight glycans

Glycans are a diverse group of biological macromolecules that are made up of polysaccharides and glycoconjugates, such as glycoproteins and glycopeptides. The different types of glycans results in a multitude of structures, properties and functions. This investigation looks at the different hydrodynamic properties of several glycans including viscosity, sedimentation coefficient and molecular weight. These parameters are determined using techniques such as viscometry and Analytical Ultracentrifugation (AUC). A foundation on the principles of the techniques involved in hydrodynamic characterisation was provided through the use of ovalbumin, a glycoprotein that has been extensively studied. The study gave a basic understanding in Analytical Ultracentrifugation. Using the knowledge obtained from that investigation a study into possible reasons why the ‘last-resort’ glycopeptide antibiotic, vancomycin, is not commonly administered orally due to poor absorption within the gut. The study examined at the interactions of vancomycin with common macromolecules found with the gastro-intestinal tract, such as mucin, and trying to explain reasons how these interactions could inhibit the absorption of vancomycin. Finally investigations into two different species derived β-glucans, that have been shown to have medically benefiting properties, were characterised. The used of different hydrodynamic techniques yielded results that generally supported the accepted hydrodynamic properties of β-glucans, however there were results that challenged these understandings as well

Characterisation of insulin analogues therapeutically available to patients

The structure and function of clinical dosage insulin and its analogues were assessed. This included ‘native insulins’ (human recombinant, bovine, porcine), ‘fast-acting analogues’ (aspart, glulisine, lispro) and ‘slow-acting analogues’ (glargine, detemir, degludec). Analytical ultracentrifugation, both sedimentation velocity and equilibrium experiments, were employed to yield distributions of both molar mass and sedimentation coefficient of all nine insulins. Size exclusion chromatography, coupled to multi-angle light scattering, was also used to explore the function of these analogues. On ultracentrifugation analysis, the insulins under investigation were found to be in numerous conformational states, however the majority of insulins were present in a primarily hexameric conformation. This was true for all native insulins and two fast-acting analogues. However, glargine was present as a dimer, detemir was a multi-hexameric system, degludec was a dodecamer (di-hexamer) and glulisine was present as a dimer-hexamer-dihexamer system. However, size-exclusion chromatography showed that the two hexameric fast-acting analogues (aspart and lispro) dissociated into monomers and dimers due to the lack of zinc in the mobile phase. This comprehensive study is the first time all nine insulins have been characterised in this way, the first time that insulin detemir have been studied using analytical ultracentrifugation and the first time that insulins aspart and glulisine have been studied using sedimentation equilibrium. The structure and function of these clinically administered insulins is of critical importance and this research adds novel data to an otherwise complex functional physiological protein

The adaptability of the ion binding site by the Ag(I)/Cu(I) periplasmic chaperone SilF

The periplasmic chaperone SilF has been identified as part of an Ag(I) detoxification system in Gram negative bacteria. Sil proteins also bind Cu(I), but with reported weaker affinity, therefore leading to the designation of a specific detoxification system for Ag(I). Using isothermal titration calorimetry we show that binding of both ions is not only tighter than previously thought, but of very similar affinities. We investigated the structural origins of ion binding using molecular dynamics and QM/MM simulations underpinned by structural and biophysical experiments. The results of this analysis showed that the binding site adapts to accommodate either ion, with key interactions with the solvent in the case of Cu(I). The implications of this are that Gram negative bacteria do not appear to have evolved a specific Ag(I) efflux system but take advantage of the existing Cu(I) detoxification system. Therefore, there are consequences for how we define a particular metal resistance mechanism and understand its evolution in the environment

c(M) vs. molar mass distributions measured using SEDFIT-MSTAR from AUC-SE data, with superimposed M*(r) extrapolations to the cell base (relative radius = 1), and c(M) fits over raw AUC-SE data in insets.

<p>(a) IHr; (b) IBov; (c) IPor; (d) IAsp; (e) IGlu; (f) ILis; (g) IGla; (h) IDet; (i) IDeg. SEDFIT-MSTAR was unable to adequately fit data from IDeg.</p

Elution from SEC plots of insulin and analogues.

<p>Black line represents PBS as the mobile phase, grey line represents TRIS. (a) IHr; (b) IBov; (c) IPor; (d) IAsp; (e) IGlu; (f) ILis; (g) IDet; (h) IDeg. IGla was not injected due to pI/pH incompatibilities. Monomers (M), Dimers (D), Hexamers (H), Dihexamers (DiH), multihexamers (multiH) and Excipients (Ex) were identified by molar mass.</p

The adaptability of the ion binding site by the Ag(I)/Cu(I) periplasmic chaperone SilF.

The periplasmic chaperone SilF has been identified as part of an Ag(I) detoxification system in Gram negative bacteria. Sil proteins also bind Cu(I), but with reported weaker affinity, therefore leading to the designation of a specific detoxification system for Ag(I). Using isothermal titration calorimetry we show that binding of both ions is not only tighter than previously thought, but of very similar affinities. We investigated the structural origins of ion binding using molecular dynamics and QM/MM simulations underpinned by structural and biophysical experiments. The results of this analysis showed that the binding site adapts to accommodate either ion, with key interactions with the solvent in the case of Cu(I). The implications of this are that Gram negative bacteria do not appear to have evolved a specific Ag(I) efflux system but take advantage of the existing Cu(I) detoxification system. Therefore, there are consequences for how we define a particular metal resistance mechanism and understand its evolution in the environment. </p

Supplementary information files for The adaptability of the ion binding site by the Ag(I)/Cu(I) periplasmic chaperone SilF.

Supplementary files for article The adaptability of the ion binding site by the Ag(I)/Cu(I) periplasmic chaperone SilF.The periplasmic chaperone SilF has been identified as part of an Ag(I) detoxification system in Gram negative bacteria. Sil proteins also bind Cu(I), but with reported weaker affinity, therefore leading to the designation of a specific detoxification system for Ag(I). Using isothermal titration calorimetry we show that binding of both ions is not only tighter than previously thought, but of very similar affinities. We investigated the structural origins of ion binding using molecular dynamics and QM/MM simulations underpinned by structural and biophysical experiments. The results of this analysis showed that the binding site adapts to accommodate either ion, with key interactions with the solvent in the case of Cu(I). The implications of this are that Gram negative bacteria do not appear to have evolved a specific Ag(I) efflux system but take advantage of the existing Cu(I) detoxification system. Therefore, there are consequences for how we define a particular metal resistance mechanism and understand its evolution in the environment.</p

Primary structure of human insulin and its analogues.

<p>Differences highlighted and numbered.</p

Molar mass distributions measured against concentration using MULTISIG-RADIUS from AUC-SE data, with superimposed n-, w- and z-average molar masses.

<p>Insets are fits from INVEQ analysis. (a) IHr; (b) IBov; (c) IPor; (d) IAsp; (e) IGlu; (f) ILis; (g) IGla; (h) IDet; (i) IDeg. MULTISIG-RADIUS was unable to adequately fit data from IDeg, but INVEQ was able.</p