Hydrodynamic modelling of protein conformation in solution: ELLIPS and HYDRO

Torre, José Garcı́a de la; Harding, Stephen E.

doi:10.1007/s12551-013-0102-6

Cited by 28 publications

(31 citation statements)

References 57 publications

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“…Like CD, AUC is another–and complementary–method for assessing protein–ligand interactions [ 43 ] and also self-association reactions [ 44 ] without the need for immobilization, labelling or an assumed inert separation matrix. It can also yield conformation information, not so much at the secondary structural level that CD can provide but at the overall gross conformational level (ellipsoids and bead models), particularly when used in conjunction with data from other techniques [ 45 ]. Although insofar as we know, it has yet to be applied to kinase-based systems, it has been applied to a number of other membrane-based systems.…”

Section: Use Of Analytical Ultracentrifugation For Oligomeric State Amentioning

confidence: 99%

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

Hussain

Harding

Hughes

et al. 2016

Biochemical Society Transactions

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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 developed at Leeds alongside Professor Steve Baldwin to whom this review is dedicated. It also reviews two 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.

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Section: Use Of Analytical Ultracentrifugation For Oligomeric State Amentioning

confidence: 99%

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

Hussain

Harding

Hughes

et al. 2016

Biochemical Society Transactions

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“…There are many possible approaches for constructing models, but Garcia de la Torre has provided the particularly useful HYDROPRO (Garcia de la Torre & Harding, 2013; Garcia De La Torre, Huertas, & Carrasco, 2000; Ortega, Amoros, & Garcia de la Torre, 2011) suite of programs to facilitate hydrodynamic bead modeling. HYDROPRO has been validated by studies of both short DNA oligonucleotides (Fernandes, Ortega, Lopez Martinez, & Garcia de la Torre, 2002) and of longer DNA polymers (Amorós, Ortega, & García de la Torre, 2011).…”

Section: Introductionmentioning

confidence: 99%

Hydrodynamic Models of G-Quadruplex Structures

Chaires

Dean

et al. 2015

Methods in Enzymology

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G-quadruplexes are noncannonical four-stranded DNA or RNA structures formed by guanine-rich repeating sequences. Guanines nucleotides can hydrogen bond to form a planar tetrad structure. Such tetrads can stack to form quadruplexes of various molecularities with a variety of types of single-stranded loops joining the tetrads. High-resolution structures may be obtained by X-ray crystallography or NMR spectroscopy for quadruplexes formed by short (≈25 nt) sequences but these methods have yet to succeed in characterizing higher-order quadruplex structures formed by longer sequences. An integrated computational and experimental approach was implemented in our laboratory to obtain structural models for higher-order quadruplexes that might form in longer telomeric or promoter sequences. In our approach atomic-level models are built using folding principles gleaned from available high-resolution structures and then optimized by molecular dynamics. The program HYDROPRO is then used to construct bead models of these structures to predict experimentally testable hydrodynamic properties. Models are validated by comparison of these properties with measured experimental values obtained by analytical ultracentrifugation or other biophysical tools. This chapter describes our approach and practical procedures.

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“…This was a ground-breaking finding, since it was believed at that time that only peptides (not polysaccharides) elicit T cell activation via MHCII antigen presentation; indeed, the work suggested a fundamental shift in the MHCII presentation paradigm (Cobb et al, 2004). The immunological events that Harding, 2013). The protein is shown to be asymmetric with an axial ratio~3.…”

Section: Zwitterionic Capsular Polysaccharidesmentioning

confidence: 97%

Glycoconjugate vaccines: some observations on carrier and production methods

MacCalman

Phillips‐Jones

Harding

2019

Biotechnology and Genetic Engineering Reviews

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Glycoconjugate vaccines use protein carriers to improve the immune response to polysaccharide antigens. The protein component allows the vaccine to interact with T cells, providing a stronger and longer-lasting immune response than a polysaccharide interacting with B cells alone. Whilst in theory the mere presence of a protein component in a vaccine should be sufficient to improve vaccine efficacy, the extent of improvement varies. In the present review, a comparison of the performances of vaccines developed with and without a protein carrier are presented. The usefulness of analytical tools for macromolecular integrity assays, in particular nuclear magnetic resonance, circular dichroism, analytical ultracentrifugation and SEC coupled to multi-angle light scattering (MALS) is indicated. Although we focus mainly on bacterial capsular polysaccharideprotein vaccines, some consideration is also given to research on experimental cancer vaccines using zwitterionic polysaccharides which, unusually for polysaccharides, are able to invoke T-cell responses and have been used in the development of potential all-polysaccharide-based cancer vaccines.A general trend of improved immunogenicity for glycoconjugate vaccines is described. Since the immunogenicity of a vaccine will also depend on carrier protein type and the way in which it has been linked to polysaccharide, the effects of different carrier proteins and production methods are also reviewed. We suggest that, in general, there is no single best carrier for use in glycoconjugate vaccines. This indicates that the choice of carrier protein is optimally made on a case-by-case basis, based on what generates the best immune response and can be produced safely in each individual case.

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Hydrodynamic modelling of protein conformation in solution: ELLIPS and HYDRO

Cited by 28 publications

References 57 publications

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

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

Hydrodynamic Models of G-Quadruplex Structures

Glycoconjugate vaccines: some observations on carrier and production methods

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