Calibration and Standardisation of Synchrotron Radiation Circular Dichroism and Conventional Circular Dichroism Spectrophotometers

Miles, Andrew J.; Wien, Frank; Lees, Jonathan G.; Rodger, Alison; Janes, Robert W.; Wallace, B.A.

doi:10.1155/2003/379137

Cited by 85 publications

(106 citation statements)

References 24 publications

(36 reference statements)

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“…The instrument was calibrated for wavelength and optical rotation using d-10-camphorsulphonic acid. 16 Samples were scanned at a temperature of 25°C using 0.01-cm path length cells at a rate of 10 nm per minute and a sample interval of 0.2 nm. Sample concentration was determined by quantitative amino acid analysis (UC Davis Core Facility, Davis, CA), and the sample concentration of the peptides was 200 M in a solution containing 25 mM HEPES buffer, pH 7.5 with 150 mM NaCl.…”

Section: Circular Dichroism Spectroscopymentioning

confidence: 99%

The N-terminus of hepcidin is essential for its interaction with ferroportin: structure-function study

Nemeth

Preza

Jung

et al. 2006

Blood

238

162

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Hepcidin is the principal iron-regulatory hormone. It acts by binding to the iron exporter ferroportin, inducing its internalization and degradation, thereby blocking cellular iron efflux. The bioactive 25 amino acid (aa) peptide has a hairpin structure stabilized by 4 disulfide bonds. We synthesized a series of hepcidin derivatives and determined their bioactivity in a cell line expressing ferroportin-GFP fusion protein, by measuring the degradation of ferroportin-GFP and the accumulation of ferritin after peptide treatment. Bioactivity was also assayed in mice by the induction of hypoferremia. Serial deletion of N-terminal amino acids caused progressive decrease in activity which was completely lost when 5 N-terminal aa's were deleted. Synthetic 3-aa and 6-aa N-terminal peptides alone, however, did not internalize ferroportin and did not interfere with ferroportin internalization by native hepcidin. Deletion of 2 C-terminal aa's did not affect peptide activity. Removal of individual disulfide bonds by pairwise substitution of cysteines with alanines also did not affect peptide activity in vitro. However, these peptides were less active in vivo, likely because of their decreased stability in circulation. G71D and K83R, substitutions previously described in humans, did not affect hepcidin activity. Apart from the essential nature of the N-terminus, hepcidin structure appears permissive for mutations. (Blood. 2006;107:328-333)

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Section: Circular Dichroism Spectroscopymentioning

confidence: 99%

The N-terminus of hepcidin is essential for its interaction with ferroportin: structure-function study

Nemeth

Preza

Jung

et al. 2006

Blood

238

162

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“…A correlation coefficient is calculated, which provides an objective means of identifying ''outlier'' spectra. An option that enables the use of a new method for improved calibration, where spectra can be standardized over the entire wavelength range using a second-order polynomial scaling procedure, is also included [18].…”

Section: Processing Functionsmentioning

confidence: 99%

CDtool—an integrated software package for circular dichroism spectroscopic data processing, analysis, and archiving

Lees

Smith

Wien

et al. 2004

Analytical Biochemistry

237

236

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CDtool is a software package written to facilitate circular dichroism (CD) spectroscopic studies on both conventional lab-based instruments and synchrotron beamlines. It takes format-independent input data from any type of CD instrument, enables a wide range of standard and advanced processing methods, and, in a single user-friendly graphics-based package, takes raw data through the entire processing procedure and, importantly, uses data-mining techniques to retain in the final output all the information associated with the processing. It permits the facile comparison of data obtained from different instruments without the need for reformatting and displays it in graphical formats suitable for publication. It also includes the ability to automatically archive the processed data. This latter feature may be especially useful in light of recent funding institution directives with regard to data sharing and archiving and requirements for ''good practice'' and ''traceability'' within the pharmaceutical industry. In addition, CDtool includes a means of interfacing with protein data bank coordinate files and calculating secondary structures from them using alternate definitions and algorithms. This feature, along with a function that permits the facile production of new reference databases, enables the creation of specialized databases for secondary structural analyses of specific types of proteins. Thus the CDtool software not only enables rapid data processing and analyses but also includes many enhanced features not available in other CD data processing/analysis packages. Ó 2004 Elsevier Inc. All rights reserved.Keywords: Circular dichroism spectroscopy; Synchrotron radiation circular dichroism (SRCD); Archive; Data processing; Software; PDB Rapid data collection is one of the advantages of using circular dichroism (CD) spectroscopy for the analysis of protein structures. It is the subsequent data processing and analysis steps that can be surprisingly time consuming. This discrepancy becomes even more acute for synchrotron radiation-based circular dichroism (SRCD) 1 spectra, which can be accumulated on a very short timescale.Currently several different software packages are required for processing, comparing, and analyzing CD data. Data collection and some processing software are usually supplied with commercial CD instruments and each SRCD beamline has developed its own data collection software. The data processing functions from the various instruments tend to differ considerably, which makes cross-instrument processing of data, and thus comparisons, extremely difficult. To surmount these problems in the past, a very simple generic program (SUPER3) was developed to include a range of processing functions [1] and simple analyses, but this software does not now adequately meet the needs of ever more sophisticated data collection procedures.Analysis tools developed for CD spectra include a wide range of secondary structure calculation algorithms [2-6] which often require reformatting, rescaling, and other man...

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“…Three baseline spectra, consisting of the corresponding flow-through from the concentration step, were averaged and subtracted from the averaged sample spectra. The resulting spectra were calibrated against a spectrum of camphoursulfonic acid collected after each synchrotron beam fill (Miles et al 2003). Data processing was carried out using CDTools software (Lees et al 2004).…”

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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Calibration and Standardisation of Synchrotron Radiation Circular Dichroism and Conventional Circular Dichroism Spectrophotometers

Cited by 85 publications

References 24 publications

The N-terminus of hepcidin is essential for its interaction with ferroportin: structure-function study

The N-terminus of hepcidin is essential for its interaction with ferroportin: structure-function study

CDtool—an integrated software package for circular dichroism spectroscopic data processing, analysis, and archiving

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

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