Spin-labeled side chains

Columbus, L., et al. (2001). Molecular motion of spin-labeled side chains in a-helices Analysis by variation of side chain structure. Biochemistry 40, 3828—3846. 

Mchaourab, H. S., et al. (1996). Motion of spin-labeled side chains in T4 lysozyme. Correlation with protein structure and dynamics. Biochemistry 35, 7692—7704. 

Abstract EPR spectroscopy of site-directed spin labeled membrane proteins is at present a common and valuable biophysical tool to study structural details and conformational transitions under conditions relevant to function. EPR is considered a complementary approach to X-ray crystallography and NMR because it provides detailed information on (1) side chain dynamics with an exquisite sensitivity for flexible regions, (2) polarity and water accessibility profiles across the membrane bilayer, and (3) distances between two spin labeled side chains during protein functioning. Despite the drawback of requiring site-directed mutagenesis for each new piece of information to be collected, EPR can be applied to any complex membrane protein system, independently of its size. This chapter describes the state of the art in the application of site-directed spin labeling (SDSL) EPR to membrane proteins, with specific focus on the different types of information which can be obtained with continuous wave and pulsed techniques. 

The structural analysis on KcsA was performed based on the mobility of each spin labeled side chain in the protein segments under investigation. It is worth recognizing in Pig. 4b that most of the CW RT spectra show multiple spectral components, characterized by different mobility (a few examples are highlighted by arrows). This is a very general property of the R1 side chain in proteins. The components reflect the anisotropy of the spin label reorientational motion, but their appearance could also have other causes. They could arise from a slow equilibrium between two different protein conformations or the presence of asymmetric sites in the protein. The molecular interpretation of different spectral components is cumbersome. Multifrequency EPR [17], temperature analysis of the CW spectra [27], pulse saturation recovery techniques [28], or high pressure EPR [29] can help unravel the possible origins of the spectral components. In the case of KcsA, the spin labels motional information was quantitatively extracted from the inverse central line width (A//q, mobility parameter) and was corroborated by the measure of the accessibility of the spin labeled side chains towards lipids (O2... 

A detailed polarity analysis can be performed on membrane proteins at cryogenic temperatures extracting the and parameters from low temperature W-band CW EPR spectra. In the temperature regime below 200 K the reorientational correlation time of an otherwise unrestricted spin label side chain exceeds 100 ns,... 

Having an accurate distance determination between the NO groups of the spin labeled side chains stUl does not directly convey the structural information at the level of the backbone of the protein which would be required for modeling structures and complexes at high resolution. To correlate the spin-spin distance constraints to the backbone-backbone distances requires modeling. At the present time, modeling approaches combining sparse accurate distance constraints to macromolecular structures are under development [7, 75]. 

SDSL EPR is sensitive to flexible regions of proteins and to dynamical changes, and can be used to measure water accessibility profiles and accurate distances between spin labeled side chains. The wealth of information that can thus be obtained makes SDSL EPR a direct tool to access conformational changes of proteins. The bridge... 

Wegener C, Savitsky A, Pfeiffer M, Mobius K, Steinhoff HJ (2001) High-field EPR-detected shifts of magnetic tensor components of spin label side chains reveal protein conformational changes the proton entrance channel of hacteriorhodopsin. Appl Magn Reson 21... 

---