Site-directed spin labeling

Introduction

Site-directed spin labeling technique is a tool for investigating protein local dynamics using electron spin resonance. The theory of SDSL is based on specific reaction of spin label with aminoacid. Spin label built in protein structure can be detected by EPR spectrometry.

SDSL is also a useful tool in examinations of protein folding process.

Spin labeling

Among the various spin labels, nitroxide SL's have enjoyed widespread use for the study of macromolecular structure and dynamics because of their stability and simple EPR signal. Nitrooxide radical is usually incorporated into a heterocyclic ring (eg pyrrolidine ) and then covalently attached to a macromolecule via linker arm. Spin label can be bound to protein by many linkers. Most common are alkyl-tiosulfonates which are Cysteine specific labels. For example MTS spin label is covalently attached to a native or egineeried Cys residue via disulfide bridge.

Spin labeling procedure

An example of spin labeling procedure:

Materials:

• Yeast iso-1-cytochrome c which posess cysteine residue;

• MTSL spin label;

• acetone;

• phosphate buffer 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 over night in 4^oC.

• Check concentration using spectrophotometric method.

• Store in -70^oC.

Spectrum