Site-directed spin labeling
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Site-directed spin labeling is a technique for investigating protein local dynamics using electron spin resonance. The theory of SDSL is based on the specific reaction of spin labels with amino acids. A spin label's built-in protein structure can be detected by EPR spectrometry.
SDSL is also a useful tool in examinations of protein folding process.
[edit] Spin labeling
Site-directed spin labeling (SDSL) was pioneered in the laboratory of Dr. W.L. Hubbell. In SDSL, sites for attachment of spin labels are introduced into recombinantly expressed proteins by site-directed mutagenesis. Functional groups contained within the spin label determine their specificity. At neutral pH, protein thiol groups specifically react with the functional groups methanethiosulfonate, maleimide, and iodoacetamide, creating a covalent bond with the amino acid Cys. Spin labels are a unique molecular reporter, in that they are paramagnetic (contain an unpaired electron). Spin labels were first synthesized in the laboratory of Dr. H.M. McConnell in 1965. Since then, a variety of nitroxide spin labels have enjoyed widespread use for the study of macromolecular structure and dynamics because of their stability and simple EPR signal. The nitroxyl radical (N-O) is usually incorporated into a heterocyclic ring (eg pyrrolidine), and the unpaired electron is predominantly localized to the N-O bond. Once incorporated into the protein, a spin label's motions are dictated by its local environment. Because spin labels are exquisitely sensitive to motion, this has profound effects on its EPR spectrum.
[edit] SDSL Research Groups
David S. Cafiso
Adrian Gross
Wayne L. Hubbell
Peter Qin
John Voss
[edit] Spin labeling procedure
An example of spin labeling procedure:
Materials:
- Yeast iso-1-cytochrome c which possess cysteine residue;
- MTSL spin label;
- acetone;
- phosphate buffer solution, pH = 7.4
Procedure:
- Mix 0.33 mg of SL with 0.025 ml acetone.
- Mix 6 mg of cytochrome c with 0.5 ml of buffer.
- Add SL solution into the protein mixture.
- Incubate for 1 hour in a dark place.
- Execute dialysis in the same buffer overnight at 4oC.
- Check concentration using spectrophotometric method.
- Store at -70oC.
