The study stating that the spin labels in the electron spin resonance (ESR) spectroscopy; Electron spin resonance spectroscopy (ESR); of spin-labeling molecules is emerging as a potent method to assess the structure and conformation of proteins and nucleic acids.
In ESR, a spin label is added to the target site using cysteine substitution. This is following by modifying the sulfhydryl group with a paramagnetic nitroxide reagent. The ESR then provides information about the mobility of nitroxide side chain, accessibility of the solvent, distance between nitroxide and other paramagnetic centers.
Novel chemistry of probes in terms of incorporation into proteins, labeling, similarity to amino acid chains, and minimal perturbation of the structure and function of proteins have led to advances in monitoring the protein backbone and microenvironment of the probe. There are two main methods of modifying the proteins using paramagnetic spin labels.
Electron spin resonance spectroscopy
One method involves attaching various nitroxides to the sulfhydryl group of the cysteine residue present in the protein to create a spin label side chain. This method requires that the presence of cysteine residues only at the desired sites, so any extra cysteines in the protein are replaced by serine or alanine. One of the most frequently used spin label is methanethiosulfonate because of its specificity and small molecular volume.
But also, the peperidine-oxyl moiety and protein backbone link are flexible; allowing the folding of proteins into native conformations. However, with this spin label, a distance distribution is obtaining; rather than a defined distance. Thus, a higher number of spin-labeling sites; and conformational searching methods are requiring for structural modeling of proteins in this case.
The suppressor methodology
In another method, a paramagnetic amino acid is incorporating into a peptide; or a protein by the method of nonsense suppressor methodology or solid phase peptide synthesis. The first method using a unique tRNA-aminoacyl tRNA synthetase pair, but very few labs can employ this method successfully. However, the solid phase synthesis of a protein with as many as 166 peptides has been achieved. Also, this method can introduce unnatural amino acids at specific sites in the target proteins.
The method used to spin label cysteine involves cysteine substitution mutagenesis. Subsequently, the protein needs to be storing in dithiothreitol (DTT) to prevent the cysteine oxidation. Before subjecting the protein to spin labeling; the protein solution is diluting with an appropriate buffer to bring the concentration down. Then, the protein solution is incubating with the spin label overnight; and the excess spin label is removing using dialysis; DEAE chromatography, Ni-NTA column, gel filtration, etc.