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

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

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

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

An example of spin labeling procedure:

Materials:

• Yeast iso-1-cytochrome c which possess cysteine residues;
• 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 4^oC.
• Check concentration using spectrophotometric method.
• Store at -70^oC.