Bifunctional Spin Labeling of Muscle Proteins: Accurate Rotational Dynamics, Orientation, and Distance by EPR

摘要: While EPR allows for the characterization of protein structure and function due to its exquisite sensitivity spin label dynamics, orientation, distance, these measurements are often limited in use labels that attached via flexible monofunctional bonds, incurring additional disorder nanosecond dynamics. In this chapter, we present methods using a bifunctional (BSL) measure muscle We demonstrate attachment eliminates internal rotation label, thereby allowing accurate measurement backbone rotational including microsecond-to-millisecond motions by saturation transfer EPR. BSL also determination helix orientation mechanically magnetically aligned systems, label's stereospecific attachment. Similarly, labeling with pair greatly enhances resolution accuracy distance measured double electron-electron resonance (DEER). Finally, when is applied high helical content an assembly orientational order (e.g., fiber or membrane), two-probe DEER experiments can be combined single-probe on oriented sample process call BEER, which has potential ab initio high-resolution determination.