Molecular flexibility of citrus pectins by combined sedimentation and viscosity analysis

The flexibility/rigidity of pectins plays an important part in their structure-function relationship and therefore on their commercial applications in the food and biomedical industries. Earlier studies based on sedimentation analysis in the ultracentrifuge have focused on molecular weight distributions and qualitative and semi-quantitative descriptions based on power law and Wales-van Holde treatments of conformation in terms of "extended" conformations [Harding, S. E., Berth, G., Ball, A., Mitchell, J.R., & Garcìa de la Torre, J. (1991). The molecular weight distribution and conformation of citrus pectins in solution studied by hydrodynamics. Carbohydrate Polymers, 168, 1-15; Morris, G. A., Foster, T. J., & Harding, S.E. (2000). The effect of degree of esterification on the hydrodynamic properties of citrus pectin. Food Hydrocolloids, 14, 227-235]. In the present study, four pectins of low degree of esterification 17-27% and one of high degree of esterification (70%) were characterised in aqueous solution (0.1 M NaCl) in terms of intrinsic viscosity [η], sedimentation coefficient (s°20,w) and weight average molar mass (Mw). Solution conformation/flexibility was estimated qualitatively using the conformation zoning method [Pavlov, G.M., Rowe, A.J., & Harding, S.E. (1997). Conformation zoning of large molecules using the analytical ultracentrifuge. Trends in Analytical Chemistry, 16, 401-405] and quantitatively (persistence length Lp) using the traditional Bohdanecky and Yamakawa-Fujii relations combined together by minimisation of a target function. Sedimentation conformation zoning showed an extended coil (Type C) conformation and persistence lengths all within the range Lp=10-13 nm (for a fixed mass per unit length)

The Solution Structures of Two Human IgG1 Antibodies Show Conformational Stability and Accommodate Their C1q and FcγR Ligands.

The human IgG1 antibody subclass shows distinct properties compared with the IgG2, IgG3, and IgG4 subclasses and is the most exploited subclass in therapeutic antibodies. It is the most abundant subclass, has a half-life as long as that of IgG2 and IgG4, binds the FcγR receptor, and activates complement. There is limited structural information on full-length human IgG1 because of the challenges of crystallization. To rectify this, we have studied the solution structures of two human IgG1 6a and 19a monoclonal antibodies in different buffers at different temperatures. Analytical ultracentrifugation showed that both antibodies were predominantly monomeric, with sedimentation coefficients s20,w (0) of 6.3-6.4 S. Only a minor dimer peak was observed, and the amount was not dependent on buffer conditions. Solution scattering showed that the x-ray radius of gyration Rg increased with salt concentration, whereas the neutron Rg values remained unchanged with temperature. The x-ray and neutron distance distribution curves P(r) revealed two peaks, M1 and M2, whose positions were unchanged in different buffers to indicate conformational stability. Constrained atomistic scattering modeling revealed predominantly asymmetric solution structures for both antibodies with extended hinge structures. Both structures were similar to the only known crystal structure of full-length human IgG1. The Fab conformations in both structures were suitably positioned to permit the Fc region to bind readily to its FcγR and C1q ligands without steric clashes, unlike human IgG4. Our molecular models for human IgG1 explain its immune activities, and we discuss its stability and function for therapeutic applications

Evaluation of diffusion coefficients by means of an approximate steady-state condition in sedimentation velocity distributions

This investigation examined the feasibility of manipulating the rotor speed in sedimentation velocity experiments to spontaneously generate an approximate steady-state condition where the extent of diffusional spreading is matched exactly by the boundary sharpening arising from negative s–c dependence. Simulated sedimentation velocity distributions based on the sedimentation characteristics for a purified mucin preparation were used to illustrate a simple procedure for determining the diffusion coefficient from such steady-state distributions in situations where the concentration dependence of the sedimentation coefficient, s = s0/(1 + Kc), was quantified in terms of the limiting sedimentation coefficient as c → 0 (s0) and the concentration coefficient (K). Those simulations established that spontaneous generation of the approximate steady state could well be a feature of sedimentation velocity distributions for many unstructured polymer systems because the requirement that Kcoω2s0/D be between 46 and 183 cm−2 is not unduly restrictive. Although spontaneous generation of the approximate steady state is also a theoretical prediction for structured macromolecular solutes exhibiting linear concentration dependence of the sedimentation coefficient, s = s0(1 − kc), the required value of k is far too large for any practical advantage to be taken of this approach with globular proteins

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

A synthesis inversion to constrain global emissions of very short‐lived chlorocarbons, dichloromethane and perchloroethylene : dichloromethane, and perchloroethylene

Dichloromethane (CH2Cl2) and perchloroethylene (C2Cl4) are chlorinated very short lived substances (Cl-VSLS) with anthropogenic sources. Recent studies highlight the increasing influence of such compounds, particularly CH2Cl2, on the stratospheric chlorine budget and therefore on ozone depletion. Here, a multiyear global-scale synthesis inversion was performed to optimize CH2Cl2 (2006–2017) and C2Cl4 (2007–2017) emissions. The approach combines long-term surface observations from global monitoring networks, output from a three-dimensional chemical transport model (TOMCAT), and novel bottom-up information on prior industry emissions. Our posterior results show an increase in global CH2Cl2 emissions from 637 ± 36 Gg yr−1 in 2006 to 1,171 ± 45 Gg yr−1 in 2017, with Asian emissions accounting for 68% and 89% of these totals, respectively. In absolute terms, Asian CH2Cl2 emissions increased annually by 51 Gg yr−1 over the study period, while European and North American emissions declined, indicating a continental-scale shift in emission distribution since the mid-2000s. For C2Cl4, we estimate a decrease in global emissions from 141 ± 14 Gg yr−1 in 2007 to 106 ± 12 Gg yr−1 in 2017. The time-varying posterior emissions offer significant improvements over the prior. Utilizing the posterior emissions leads to modeled tropospheric CH2Cl2 and C2Cl4 abundances and trends in good agreement to those observed (including independent observations to the inversion). A shorter C2Cl4 lifetime, from including an uncertain Cl sink, leads to larger global C2Cl4 emissions by a factor of ~1.5, which in some places improves model-measurement agreement. The sensitivity of our findings to assumptions in the inversion procedure, including CH2Cl2 oceanic emissions, is discussed

Purification of bacterial membrane sensor kinases and biophysical methods for determination of their ligand and inhibitor interactions

This article reviews current methods for the reliable heterologous overexpression in Escherichia coli and purification of milligram quantities of bacterial membrane sensor kinase (MSK) proteins belonging to the two-component signal transduction family of integral membrane proteins. Many of these methods were developedatLeedsalongsideProfessor SteveBaldwintowhomthisreviewisdedicated.Italsoreviewstwo biophysical methods that we have adapted successfully for studies of purified MSKs and other membrane proteins – synchrotron radiation circular dichroism (SRCD) spectroscopy and analytical ultracentrifugation (AUC), both of which are non-immobilization and matrix-free methods that require no labelling strategies. Other techniques such as isothermal titration calorimetry (ITC) also share these features but generally require high concentrations of material. In common with many other biophysical techniques, both of these biophysical methods provide information regarding membrane protein conformation, oligomerization state and ligand binding, but they possess the additional advantage of providing direct assessments of whether ligand binding interactions are accompanied by conformational changes. Therefore, both methods provide a powerful means by which to identify and characterize inhibitor binding and any associated protein conformational changes, thereby contributing valuable information for future drug intervention strategies directed towards bacterial MSKs

Assessing sedimentation equilibrium profiles in analytical ultracentrifugation experiments on macromolecules: from simple average molecular weight analysis to molecular weight distribution and interaction analysis

Molecular weights (molar masses), molecular weight distributions, dissociation constants and other interaction parameters are fundamental characteristics of proteins, nucleic acids, polysaccharides and glycoconjugates in solution. Sedimentation equilibrium in the analytical ultracentrifugation provides a powerful method with no supplementary immobilization, columns or membranes required. It is particularly powerful when used in conjunction with its sister technique, namely sedimentation velocity analysis. We describe key approaches now available and their application to the characterisation of antibodies polysaccharides and glycoconjugates. We indicate how major complications such as thermodynamic non-ideality can now be routinely dealt with, thanks to a great extent to the extensive contribution of Professor DonWinzor over several decades of research

Controlled depolymerisation, as assessed by analytical ultracentrifugation, of low molecular weight chitosan for potential use in archaeological conservation

The heterogeneity and molecular weight of a chitosan of low molecular weight (molar mass) and low degree of acetylation (0.1), for potential use as a consolidant for decayed archaeological wood, has been examined by sedimentation velocity and sedimentation equilibriumin the analytical ultracentrifuge before and after depolymerisation. Sedimentation velocity before polymerisation revealed a uniform distribution of sedimentation coefficient with little concentration dependence. SEDFIT-MSTAR analysis revealed a weight average molecular weight Mw of (14.2 + 1.2) kDa, and polydispersity index of ~ 1.2. Further analysis using MULTISIG revealed a distribution of material between 2-20 kDa and consistent with the weight average Mw. Controlled depolymerisation using hydrogen peroxide and UV in an acetic acid medium reduced this to (4.9 + 0.7) kDa, with a similar polydispersity. The depolymerised material appears to be within the range that has been predicted to fully penetrate into archaeological wood. The consequences for this and the use of the analytical ultracentrifuge in wood conservation strategies is considered