Abstract Tomato bushy stunt virus particles have previously been physically and chemically well characterized. Different strains, however, are known to have different host plants and symptoms, and they can be distinguished serologically. In an attempt to correlate this behaviour with physicochemical properties, we have performed a comparative study on isolates of four of the main variants of this virus (Type strain, petunia asteroid mosaic virus, pelargonium leaf curl virus, and carnation Italian ringspot virus) with regard to sedimentation velocity characteristics, translation diffusivity (from quasi‐elastic light scattering measurements), and molecular weight (from both low‐speed sedimentation equilibrium and the Svedberg equation). It is demonstrated that there are no clear differences between the four strains, particularly insofar as their sedimentation characteristics (both velocity and low‐speed equilibrium). There is some suggestion from diffusion measurements that the Type strain virus particles may have a higher mass, although the presence of some aggregation phenomena may provide an alternative explanation for the diffusion data.
Part 1 Techniques: static and dynamic light scattering approaches to structure determination of biopolymers the use of low angle laser light scattering with gel permeation chromatography for the molecular weight determination of biomolecules combined differential light scattering with various liquid chromatography separation techniques new tools of biochemists - combined laser doppler microelectrophoresis and photon correlation spectroscopy an on-line dynamic scattering instrument for macromolecular characterization an overview of current methods of analyzing QLS data low-bias macroscopic analysis of polydispersity mesoparticle diffusion of biopolymer and polymer solutions the diffusion of BSA molecules on poly(N-isopropyl acrylamide) gels - a forced Rayleigh scattering study developments in electrophoretic laser light scattering and some biochemical applications an investigation of rigid rod-like particles in dilute solution static and dynamic light scattering by semi-dilute rod-like polyelectrolytes quasi-elastic light scattering from an evanescent wave to probe particle/wall interactions. Part 2 Macromolecules: compactness of protein in molecules in native and denatured states as revealed by laser light scattering and X-ray scattering dynamic light scattering studies of concentrated Casein Micelle suspensions aggregation of proteins hydrodynamic behaviour of myosin filaments studied by laser light scattering quasielastic and total intensity light scattering studies of mucin glycoproteins and cartilage proteoglycans use of on-line laser light scattering coupled to chromatographic separations for the determination of molecular weight, branching, size and shape distributions of polysaccharides chain rigidity of polyuronates - static light scattering of aqueous solutions of hyaluronate and alginate laser light scattering studies of polysaccharide gels light scattering studies of DNA condensation dynamics and structures of DNA DNAs as models of rigid and semi-rigid rod-like macromolecules. Part 3 Macromolecular assemblies: total intensity and quasi-elastic light scattering applications in microbiology.
The chaperonin activity of sequence-related chaperonin 10 proteins requires their aggregation into heptameric structures. We describe size-exclusion chromatography and ultracentrifugation studies that reveal that while Escherichia coli chaperonin 10 exists as a heptamer, the Mycobacterium tuberculosis chaperonin 10 is tetrameric in dilute solutions and in whole M. tuberculosis lysate. At high protein concentration and in the presence of saturating amounts of divalent ions, the protein is heptameric. Human chaperonin 10 is predominantly heptameric, although smaller oligomers were detected. These differences in structural assembly between species may explain differences in biological activity such as antigenicity.Using C-terminal and N-terminal fragments, sequence 1-25 was identified as indispensable for aggregation. CD spectroscopy studies revealed that (i) a minimum at 202-204 nm correlates with aggregation and characterizes not only the spectrum of the mycobacterial protein, but also those of E. coli and human chaperonin 10 proteins; (ii) the interactions between subunits are of the hydrophobic type; and (iii) the anti-parallel β-pleated sheet is the main secondary structure element of subunits in both tetrameric and heptameric proteins. The chaperonin activity of sequence-related chaperonin 10 proteins requires their aggregation into heptameric structures. We describe size-exclusion chromatography and ultracentrifugation studies that reveal that while Escherichia coli chaperonin 10 exists as a heptamer, the Mycobacterium tuberculosis chaperonin 10 is tetrameric in dilute solutions and in whole M. tuberculosis lysate. At high protein concentration and in the presence of saturating amounts of divalent ions, the protein is heptameric. Human chaperonin 10 is predominantly heptameric, although smaller oligomers were detected. These differences in structural assembly between species may explain differences in biological activity such as antigenicity. Using C-terminal and N-terminal fragments, sequence 1-25 was identified as indispensable for aggregation. CD spectroscopy studies revealed that (i) a minimum at 202-204 nm correlates with aggregation and characterizes not only the spectrum of the mycobacterial protein, but also those of E. coli and human chaperonin 10 proteins; (ii) the interactions between subunits are of the hydrophobic type; and (iii) the anti-parallel β-pleated sheet is the main secondary structure element of subunits in both tetrameric and heptameric proteins.
In establishing the sources of data variability within sedimentation velocity analysis in the analytical ultracentrifuge and their relative importance, recent studies have demonstrated that alignment of the sample cells to the centre of rotation is the most significant contributing factor to overall variability, particularly for the characterisation of low levels of protein aggregation. Accurate mechanical and optical alignment tools have been recently designed. In this study, we (1) confirm the effect of misalignment observed by others on the estimated amounts of bovine serum albumin (BSA) monomer and dimer, and the sedimentation coefficient value for the BSA dimer; and (2) demonstrate the high performance of a mechanical alignment tool and the usefulness of a simple and complementary enhanced manual alignment protocol which should be useful for situations where these tools are not available.
Analytical ultracentrifugation is a versatile approach for analysing the molecular mass, molecular integrity (degradation/aggregation), oligomeric state and association/dissociation constants for self-association, and assay of ligand binding of kinase related membrane proteins and glycans. It has the great property of being matrix free-providing separation and analysis of macromolecular species without the need of a separation matrix or membrane or immobilisation onto a surface. This short review-designed for the non-hydrodynamic expert-examines the potential of modern sedimentation velocity and sedimentation equilibrium and the challenges posed for these molecules particularly those which have significant cytoplasmic or extracellular domains in addition to the transmembrane region. These different regions can generate different optimal requirements in terms of choice of the appropriate solvent (aqueous/detergent). We compare how analytical ultracentrifugation has contributed to our understanding of two kinase related cellular or bacterial protein/glycan systems (i) the membrane erythrocyte band 3 protein system-studied in aqueous and detergent based solvent systems-and (ii) what it has contributed so far to our understanding of the enterococcal VanS, the glycan ligand vancomycin and interactions of vancomycin with mucins from the gastrointestinal tract.
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