Chemical Sciences: A Manual for CSIR-UGC National Eligibility Test for Lectureship and JRF/Site-directed spin labeling

From Wikibooks, open books for an open world
Jump to navigation Jump to search

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.

Spin labeling[edit]

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 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 (e.g. 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.

SDSL Research Groups[edit]

Albert H. Beth
David S. Cafiso
Jack Freed
Betty J. Gaffney
Adrian Gross
Wayne L. Hubbell
Heinz-Jürgen Steinhoff
Ralf Langen
Gary Lorigan
Peter Qin
Linda L. Randall
David D. Thomas
John Voss
Gail E. Fanucci

File:Epr spectrum.jpg
EPR spectrum of proxyl-MTS spin labeled yeast iso-1-cytochrome c. Spin label is attached to Cysteine 102 residue.

Spin labeling procedure[edit]

An example of spin labeling procedure:

Materials:

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.