Site-directed spin labeling (SDSL) is a technique for investigating the structure and local dynamics of

protein Proteins are large biomolecules and macromolecules that comprise one or more long chains of amino acid residues. Proteins perform a vast array of functions within organisms, including catalysing metabolic reactions, DNA replication, respon ...

s using

electron spin resonance Electron paramagnetic resonance (EPR) or electron spin resonance (ESR) spectroscopy is a method for studying materials that have unpaired electrons. The basic concepts of EPR are analogous to those of nuclear magnetic resonance (NMR), but the s ...

. The theory of SDSL is based on the specific reaction of

spin label A spin label (SL) is an organic molecule which possesses an unpaired electron, usually on a nitrogen atom, and the ability to bind to another molecule. Spin labels are normally used as tools for probing proteins or biological membrane-local dynami ...

s with

amino acid Amino acids are organic compounds that contain both amino and carboxylic acid functional groups. Although hundreds of amino acids exist in nature, by far the most important are the alpha-amino acids, which comprise proteins. Only 22 alpha ...

s. A spin label's built-in protein structure can be detected by EPR spectroscopy. SDSL is also a useful tool in examinations of the

protein folding Protein folding is the physical process by which a protein chain is translated to its native three-dimensional structure, typically a "folded" conformation by which the protein becomes biologically functional. Via an expeditious and reprodu ...

process.

Spin labeling

Site-directed spin labeling (SDSL) was pioneered in the laboratory of

Dr. W.L. Hubbell Wayne L. Hubbell (born 24 March 1943) is an American biochemist and member of the National Academy of Sciences. He is Professor of Biochemistry and Jules Stein Professor of Ophthalmology at the University of California, Los Angeles. His rese ...

. In SDSL, sites for attachment of spin labels are introduced into recombinantly expressed proteins by site-directed mutagenesis.

Functional group In organic chemistry, a functional group is a substituent or moiety in a molecule that causes the molecule's characteristic chemical reactions. The same functional group will undergo the same or similar chemical reactions regardless of the res ...

s 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 Cysteine (symbol Cys or C; ) is a semiessential proteinogenic amino acid with the formula . The thiol side chain in cysteine often participates in enzymatic reactions as a nucleophile. When present as a deprotonated catalytic residue, som ...

. 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 Harden M. McConnell (July 18, 1927 – October 8, 2014) was an American physical chemist. His many awards included the National Medal of Science and the Wolf Prize, and he was elected to the National Academy of Science." Education and career Hard ...

in 1965. Since then, a variety of nitroxide

s 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 Pyrrolidine, also known as tetrahydropyrrole, is an organic compound with the molecular formula (CH2)4NH. It is a cyclic secondary amine, also classified as a saturated heterocycle. It is a colourless liquid that is miscible with water and most ...

), 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. The assembly of multi-subunit membrane protein complexes has also been studied using spin labeling. The binding of the PsaC subunit to the PsaA and PsaB subunits of the photosynthetic reaction center, Photosystem I, has been analyzed in great detail using this technique. Dr. Ralf Langen's group showed that SDSL with EPR (University of Southern California, Los Angeles) can be used to understand the structure of amyloid fibrils and the structure of membrane bound Parkinson's disease protein alpha-synuclein. A 2012 study generated a high resolution structure of IAPP fibrils using a combination of SDSL, pulse EPR and computational biology.

References

{{DEFAULTSORT:Site-Directed Spin Labeling Analytical chemistry Spectroscopy Protein methods