Please use this identifier to cite or link to this item: http://hdl.handle.net/1959.14/163953
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- Title
- Distance measurements by continuous wave EPR spectroscopy to monitor protein folding
- Related
- Hill, Andrew F.; Barnham, Kevin J.; Bottomley, Stephen P. and Cappai, Roberto. Protein folding, misfolding, and disease : methods and protocols, p.73-96
- DOI
- 10.1007/978-1-60327-223-0_6
- Related
- Methods in molecular biology Vol. 752
- Publisher
- New York, N.Y : Humana
- Date
- 2011
- Author/Creator
- Cooke, James A
- Author/Creator
- Brown, Louise J
- Description
- Site-Directed Spin Labeling Electron Paramagnetic Resonance (SDSL-EPR) offers a powerful method for the structural analysis of protein folds. This method can be used to test and build secondary, tertiary, and quaternary structural models as well as measure protein conformational changes in solution. Insertion of two cysteine residues into the protein backbone using molecular biology methods and the subsequent labeling of the cysteine residues with a paramagnetic spin label enables the technique of EPR to be used as a molecular spectroscopic ruler. EPR measures the dipolar interaction between pairs of paramagnetic spin labels to yield internitroxide distances from which quantitative structural information on a protein fold can then be obtained. Interspin dipolar interaction between two spin labels at less than 25 Å are measured using continuous wave (CW) EPR methods. As for any low-resolution distance methods, the positioning of the spin labels and the number of distance constraints to be measured are dependent on the structural question being asked, thus a pattern approach for using distance sets to decipher structure mapping, including protein folds and conformational changes associated with biological activity, is essential. Practical guidelines and hints for the technique of SDSL-EPR are described in this chapter, including methods for spin labeling the protein backbone, CW-EPR data collection at physiological temperatures and two semiquantitative analysis methods to extract interspin distance information from the CW-EPR spectra.
- Description
- 24 pages(s)
- Subject Keyword
- Site-directed spin labeling
- Subject Keyword
- Electron paramagnetic resonance
- Subject Keyword
- Protein folding
- Subject Keyword
- Continuous wave EPR spectroscopy
- Subject Keyword
- Site-directed mutagenesis
- Subject Keyword
- Interspin distances
- Subject Keyword
- Dipolar interaction
- Resource Type
- book chapter
- Organisation
- Macquarie University. Dept. of Chemistry and Biomolecular Sciences
- Identifier
- http://hdl.handle.net/1959.14/163953
- Identifier
- ISBN:9781603272216
- Identifier
- ISSN:1064-3745
- Identifier
- mq_res-20120329-160112
- Language
- eng
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