A reference dataset for the analyses of membrane protein secondary structures and transmembrane residues
using circular dichroism spectroscopy

Abdul-Gader, Ali; Miles, Andrew J.; Wallace, B.A.

doi:10.1093/bioinformatics/btr234

Cited by 144 publications

(140 citation statements)

References 40 publications

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“…The measurements were performed at a scanning speed of 50 nm/min, a response time of 2 seconds, a bandwidth of 1 nm, a sensitivity of 100 m, steps of 0·5 nm, and an accumulation of six scans. Data were analysed using the spectra analysis software dichroweb (University of London, London, UK) and reference data set SMP180 24. Data were converted to mean residue ellipticity by the software using a mean amino acid residue mass of 113.…”

Section: Methodsmentioning

confidence: 99%

Influence of Escherichia coli chaperone DnaK on protein immunogenicity

et al. 2016

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SummaryThe production of anti‐drug antibodies can impact significantly upon the safety and efficacy of biotherapeutics. It is known that various factors, including aggregation and the presence of process‐related impurities, can modify and augment the immunogenic potential of proteins. The purpose of the investigations reported here was to characterize in mice the influence of aggregation and host cell protein impurities on the immunogenicity of a humanized single‐chain antibody variable fragment (scFv), and mouse albumin. Host cell protein impurities within an scFv preparation purified from Escherichia coli displayed adjuvant‐like activity for responses to the scFv in BALB/c strain mice. The 70 000 MW E. coli chaperone protein DnaK was identified as a key contaminant of scFv by mass spectrometric analysis. Preparations of scFv lacking detectable DnaK were spiked with recombinant E. coli DnaK to mimic the process‐related impurity. Mice were immunized with monomeric and aggregated preparations, with and without 0·1% DnaK by mass. Aggregation alone enhanced IgM and IgG2a antibody responses, but had no significant effect on total IgG or IgG1 responses. The addition of DnaK further enhanced IgG and IgG2a antibody responses, but only in the presence of aggregated protein. DnaK was shown to be associated with the aggregated scFv by Western blot analysis. Experiments with mouse albumin showed an overall increase in immunogenicity with protein aggregation alone, and the presence of DnaK increased the vigour of the IgG2a antibody response further. Collectively these data reveal that DnaK has the potential to modify and enhance immunogenicity when associated with aggregated protein.

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Section: Methodsmentioning

confidence: 99%

Influence of Escherichia coli chaperone DnaK on protein immunogenicity

et al. 2016

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“…Data processing was carried out using CDTools software (Lees et al 2004). The spectra were truncated to 180 nm (the wavelength indicated by the HT cutoff criterion (Miles and Wallace, 2006), smoothed with a SavitskyGolay filter, and scaled to delta epsilon values using mean residue weight values calculated for each construct (114.3, 113.9, 113.4, 12.7, 112.5, and 112.6 for full length, for 277, Δ265, Δ259, Δ250, Δ239, and Δ234, respectively, and analyses used the DichroWeb analysis server (14) with the CONTINLL algorithm (Provencher and Glöckner 1981;Van Stokkum et al 1990), and the MP180 reference set (Abdul-Gader et al 2011). …”

Section: Synchrotron Radiation Circular Dichroism (Srcd) Spectroscopymentioning

confidence: 99%

Structure of the C-terminal domain of the prokaryotic sodium channel orthologue NsvBa

2016

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Crystallographic and electrophysiological studies have recently provided insight into the structure, function and drug binding of prokaryotic sodium channels. These channels exhibit significant sequence identities, especially in their transmembrane regions, with human voltage-gated sodium channels. However, rather than being single polypeptides with four homologous domains, they are tetramers of single domain polypeptides, with a C-terminal domain (CTD) composed of an inter-subunit four helix coiled-coil. The structures of the CTDs differ between orthologues. In NavBh and NavMs, the C-termini form a disordered region adjacent to the final transmembrane helix, followed by a coiled-coil region, as demonstrated by synchrotron radiation circular dichroism (SRCD) and double electronelectron resonance electron paramagnetic resonance spectroscopic measurements. In contrast, in the crystal structure of the NavAe orthologue, the entire C-terminus is comprised of a helical region followed by a coiled-coil. In this study we have examined the CTD of the NsvBa from Bacillus alcalophilus, which unlike other orthologues is predicted by different methods to have different types of structures: either a disordered adjacent to the transmembrane region, followed by a helical coiled-coil, or a fully helical CTD. To discriminate between the two possible structures we have used SRCD spectroscopy to experimentally determine the secondary structure of the C-terminus of this orthologue and used the results as the basis for modelling the transition between open and closed conformations of the channel.3

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“…Secondary structure analyses were carried out using the DichroWeb analysis server [19]. Values from the algorithms CONTINLL [20,21], SELCON [22] and CDDSTR [22] (using reference set SMP180 [23]) were averaged and their standard deviations reported. SMP180 is a new reference data set specifically devised for use with membrane proteins.…”

Section: Srcd Spectroscopymentioning

confidence: 99%

“…3C and F). a Values from the algorithms CONTINLL [20,21], SELCON [22] and CDDSTR [22] (using reference set SMP180 [23]). The values reported are the average of the three algorithms, with ± reflecting the standard deviation between the values calculated by the different methods.…”

Section: Spectral Analyses Of Thermaldenaturation Of Solubilised Nakmentioning

confidence: 99%

Stabilisation of Na,K-ATPase structure by the cardiotonic steroid ouabain

Miles

Fedosova

Hoffmann

et al. 2013

Biochemical and Biophysical Research Communications

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a b s t r a c tCardiotonic steroids such as ouabain bind with high affinity to the membrane-bound cation-transporting P-type Na,K-ATPase, leading to complete inhibition of the enzyme. Using synchrotron radiation circular dichroism spectroscopy we show that the enzyme-ouabain complex is less susceptible to thermal denaturation (unfolding) than the ouabain-free enzyme, and this protection is observed with Na,K-ATPase purified from pig kidney as well as from shark rectal glands. It is also shown that detergent-solubilised preparations of Na,K-ATPase are stabilised by ouabain, which could account for the successful crystallisation of Na,K-ATPase in the ouabain-bound form. The secondary structure is not significantly affected by the binding of ouabain. Ouabain appears however, to induce a reorganization of the tertiary structure towards a more compact protein structure which is less prone to unfolding; recent crystal structures of the two enzymes are consistent with this interpretation. These circular dichroism spectroscopic studies in solution therefore provide complementary information to that provided by crystallography.

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A reference dataset for the analyses of membrane protein secondary structures and transmembrane residues using circular dichroism spectroscopy

Abstract: Reference dataset online at the DichroWeb analysis server (http://dichroweb.cryst.bbk.ac.uk); individual protein spectra in the Protein Circular Dichroism Data Bank (http://pcddb.cryst.bbk.ac.uk).

Cited by 144 publications

References 40 publications

Influence of Escherichia coli chaperone DnaK on protein immunogenicity

Influence of Escherichia coli chaperone DnaK on protein immunogenicity

Structure of the C-terminal domain of the prokaryotic sodium channel orthologue NsvBa

Stabilisation of Na,K-ATPase structure by the cardiotonic steroid ouabain

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