Electron Spin Resonance Spectroscopy Labeling in Peptide and Protein Analysis

Electron spin resonance (ESR) is a powerful analytical tool used in protein and peptide biochemistry. It is used in the determination of secondary, tertiary and quaternary protein structure and associated conformational changes. Protein dynamics and the relative orientation of protein components in ordered systems can also be measured. The majority of proteins do not contain unpaired electrons whose spin transitions give rise to an ESR signal, hence necessitating the use of extrinsic probes called spin labels. Spin labels are nitroxide derivatives with a stable unpaired electron and a functional group for specific attachment to the protein (covalent or as a ligand). The most popular covalent sites are cysteine residues, which, if necessary, can be introduced into the protein structure using molecular biology techniques. The physical basis for nearly all ESR applications is the anisotropy of the nitroxide signal and the sensitivity of the ESR spectra to various relaxation pathways. The interaction between an electron of a spin label and an external magnetic field depends on their relative orientations. The splitting and the center of ESR spectra of an oriented sample are used to determine the orientation of labeled domains. For samples with little disorder the orientational sensitivity is better than 1°. The width of the signal is proportional to the orientational disorder, which is used to measure conformational heterogeneity of proteins. If the spin label reorientates itself on the ESR timescale (nanoseconds) then the spectral anisotropy is averaged. The extent of averaging defines the ESR line shape which is used to determine the rotational rate and anisotropy of motion. The dynamic range of ESR is very broad, rotational correlation times range from 10−12 to 10−7 s for conventional ESR and the sensitivity can be extended to slower motions (10−3 s) with nonlinear saturation transfer electron spin resonance (STESR). Protein (spin label) mobility is used to follow conformational changes, steric restrictions on the spin label and the formation of large complexes. Spin labels are also sensitive to the presence of other paramagnetic species. Collisions with water and lipid-soluble relaxing agents provide additional relaxation pathways measured by changes in relaxation times. The probability of these collisions reflects the accessibility of a spin label to the relaxant. The periodic patterns along the polypeptide chain of this accessibility are used to determine the secondary and tertiary structure of proteins. In the presence of another bound spin label or a paramagnetic metal complexed by histidine residues, spectra become broadened by dipolar or exchange interactions. Both mechanisms depend on the distance between the paramagnetic centers. Thus ESR can be used to determine intra- and intermolecular distances. The range of sensitivity is 5–25 A and there are intensive efforts to increase the upper range to >50 A. ESR as a spectroscopic ruler is used in protein structure determination and the investigation of macromolecular assembly processes and protein folding. The foremost limitation of spin labeling ESR is the necessity to modify a protein with a spin probe. In some cases, the spin labels may perturb protein function and therefore cannot be used for spectroscopy. However, even an unsuccessful modification that results in functional loss identifies functional regions of proteins and as such represents successful “mutational analysis” experiments.