DICHROWEB, an online server for protein secondary structure analyses from circular dichroism spectroscopic data

Whitmore, Lee; Wallace, B.A.

doi:10.1093/nar/gkh371

Cited by 2,229 publications

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“…For secondary structure predictions, spectra in the far-UV region (190-240 nm) were recorded at 22 C with 0.5 nm increments in a 0.5 mm quartz cell, and six scans were averaged for each spectrum. Secondary structure estimates of wild type and mutant MexR was made using DICHROWEB 33,51 and the algorithms CDSSTR, 52,53 CONTIN, 54,55 and Selcon3. 56 Thermal denaturation spectra were recorded at 210-240 nm with 5 C intervals from 15-85 C using a 0.5 mm quartz cell, 15 lM protein in 10 mM sodium phosphate buffer, pH 7.2.…”

Section: Circular Dichroism Experimentsmentioning

confidence: 99%

Critical biophysical properties in the Pseudomonas aeruginosa efflux gene regulator MexR are targeted by mutations conferring multidrug resistance

et al. 2010

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The self-assembling MexA-MexB-OprM efflux pump system, encoded by the mexO operon, contributes to facile resistance of Pseudomonas aeruginosa by actively extruding multiple antimicrobials. MexR negatively regulates the mexO operon, comprising two adjacent MexR binding sites, and is as such highly targeted by mutations that confer multidrug resistance (MDR). To understand how MDR mutations impair MexR function, we studied MexR-wt as well as a selected set of MDR single mutants distant from the proposed DNA-binding helix. Although DNA affinity and MexA-MexB-OprM repression were both drastically impaired in the selected MexR-MDR mutants, MexR-wt bound its two binding sites in the mexO with high affinity as a dimer. In the MexR-MDR mutants, secondary structure content and oligomerization properties were very similar to MexR-wt despite their lack of DNA binding. Despite this, the MexR-MDR mutants showed highly varying stabilities compared with MexR-wt, suggesting disturbed critical interdomain contacts, because mutations in the DNA-binding domains affected the stability of the dimer region and vice versa. Furthermore, significant ANS binding to MexR-wt in both free and DNA-bound states, together with increased ANS binding in all studied mutants, suggest that a hydrophobic cavity in the dimer region already shown to be involved in regulatory binding is enlarged by MDR mutations. Taken together, we propose that the biophysical MexR properties that are targeted by MDR mutations-stability, domain interactions, and internal hydrophobic surfaces-are also critical for the regulation of MexR DNA binding.

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Section: Circular Dichroism Experimentsmentioning

confidence: 99%

Critical biophysical properties in the Pseudomonas aeruginosa efflux gene regulator MexR are targeted by mutations conferring multidrug resistance

et al. 2010

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“…The spectra of the target fragment in all four environments showed two minima at near 208 nm and 222 nm, typical for α-helical secondary structure. The raw CD data were converted to mean residue molar ellipticity [θ] and the secondary structure content of the peptide was analyzed by using the program ContinLL on DICHROWEB, an interactive web site allowing the deconvolution of data from circular dichroism spectroscopy experiments [22,23]. The results showed that CB2 271-326 adopts high α-helical structure in the all of the environments.…”

Section: Refolding and Secondary Structure Of Cb2 271-326 In Differenmentioning

confidence: 99%

“…The resulting spectra were analyzed for the proportions of various secondary structures using the computer program ContinLL supported by an interactive web site DICHROWEB [22,23].…”

Section: Circular Dichroismmentioning

confidence: 99%

“…The peptide was solubilized in 4 mM digitonin containing 10 mM sodium phosphate buffer, pH 7.0, 50 mM NaCl. The CD spectra taken in four different environments were analyzed by the program ContinLL [22,23].…”

Section: Stern-volmer Quenchingmentioning

confidence: 99%

“…After brief centrifugation (13,000 rpm, 5 min), the supernatant was carefully transferred to a new tube and the protein concentration was determined by UV spectrometry using extinction This calculated concentration was used to convert the CD spectra from CD mdeg to mean residue ellipticity [θ]. The resulting spectra were analyzed for the proportions of various secondary structures using the computer program ContinLL supported by an interactive web site DICHROWEB [22,23]. …”

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Biosynthesis, purification, and characterization of a cannabinoid receptor 2 fragment (CB2271–326)

Zhang

Xie

2008

Protein Expression and Purification

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Obtaining sufficient amount of purified G-protein coupled receptors (GPCRs) is almost always one of the major challenges for their structural studies. CB2 , a human cannabinoid receptor 2 (CB2) fragment comprising part of the third extracellular loop (EL3), the seventh transmembrane domain (TM7) and C-terminal juxtamembrane region of the receptor, was over-expressed as a fusion protein into inclusion body (IB) of Escherichia coli. The fusion protein was purified by histidineselected nickel affinity chromatography under denaturing conditions. Then, the fusion protein IBs were solubilized in detergent (Brij58) and the expression fusion leader sequence (TrpLE) was specifically cleaved with tobacco etch virus (TEV) protease. The target fragment, CB2 , was subsequently purified by reverse-phase HPLC and confirmed by SDS-PAGE and mass spectrometry. This hydrophobic fragment can refold in mild detergents digitonin and Brij58. Circular dichroism (CD) spectroscopy of CB2 in digitonin and Brij58 micelles showed that the fragment adopts a more than 75% α-helical structure, with the remainder having β-strand structure. Fluorescence spectroscopy and quenching studies suggested that the C-terminal region lies near the surface of the digitonin micelles and the TM7 region is folded relatively close to the center of the micelles. This study may provide an alternative strategy for the production and structure/functional studies of GPCRs such as CB2 receptor protein produced in the form of IBs. KeywordsCannabinoid receptor 2; transmembrane domain; inclusion body; affinity chromatography; HPLC; circular dichroism; fluorescence Since the discovery of the CB2 receptor in 1993 [1], there has been a growing interest to clarify the importance of this GPCR membrane protein in human physiology, and to investigate it as a possible target for current and future drug development. The CB2 receptor seems to have a significant role in mediating the immunological functions of cannabinoid receptor ligands. Thus, the CB2 receptor is a promising target for drug development that aims at management of neurological pain, inflammation, osteoporosis, and growth of malignant gliomas and tumors * Corresponding author. Fax: +1 412 383 5276. E-mail address: xix15@pitt.edu (X-Q. Xie). Publisher's Disclaimer: This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final citable form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. [3][4][5][6][7]. In general, the Escherichia coli expression system is the most attractive strategy due to its advantages of simplicity of use, low cost, easy scale-up, and homogeneous protein production. Though few attempts were promising to express full length CB2 receptor in ...

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Circular Dichroism to Study Protein Interactions

Kelly

Price

2006

CP Protein Science

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Circular dichroism (CD) is a powerful technique for studying the structures of proteins in solution, as well as structural changes that may occur when proteins bind to ligands. Changes in CD signals accompanying complex formation can be used to determine the strength of the interaction and to provide information about the nature and extent of the conformational changes involved. This unit outlines the type of information available from CD studies and describes how such experiments should be carried out to ensure that reliable information is obtained.

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DICHROWEB, an online server for protein secondary structure analyses from circular dichroism spectroscopic data

Cited by 2,229 publications

References 20 publications

Critical biophysical properties in the Pseudomonas aeruginosa efflux gene regulator MexR are targeted by mutations conferring multidrug resistance

Critical biophysical properties in the Pseudomonas aeruginosa efflux gene regulator MexR are targeted by mutations conferring multidrug resistance

Biosynthesis, purification, and characterization of a cannabinoid receptor 2 fragment (CB2271–326)

Circular Dichroism to Study Protein Interactions

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