Structural Information from CW-EPR Spectra of Dipolar Coupled Nitroxide Spin Labels

Abstract

Abstract:Site directed spin labeling has emerged as a powerful technique for determining structural features of proteins. By incorporating two spin labeled side chains, distances between elements of secondary structure can be determined by quantitation of spin-spin interactions between the probes. Recent advances in methods for extracting both the distance between spin labeled probes and their relative orientations are providing the capability for testing and refining accurate structural models for a wide variety of proteins. These methods take advantage of multifrequency EPR and global non-linear data analysis tools. The capabilities of current analytical methods are described for three relevant models of spin-spin interactions and directions for further refinement of these methods for future applications are described. Reliable structural information can be obtained for interelectron distances ranging from 5 to 20 A and up to 25 A using perdeuterated probes.

A Straightforward Approach to the Analysis of Double Electron–Electron Resonance Data

“…Electron–electron dipolar coupling will produce significant broadening of continuous wave EPR lineshapes at distances up to 20 Å at X-band (for reviews see Hustedt & Beth, 1999; Hustedt & Beth, 2000; Persson et al, 2001) and even longer distances at lower frequencies (Kittell, Hustedt & Hyde, 2012). Methods have been developed for extracting an interspin distance, or distribution of distances, from these spectra (Rabenstein & Shin, 1995; Hustedt, Smirnov, Laub, Cobb & Beth, 1997; Hustedt, Stein, Sethaphong, Brandon, Zhou & Desensi, 2006).…”

“…A great deal of site-specific information including spin-label side-chain dynamics and solvent accessibility can be obtained from conventional continuous wave EPR measurements on singly-labeled molecules. In addition, it is also possible to obtain inter-probe distance and distance distribution information in the 5 to 20 Å range from continuous wave EPR measurements (Rabenstein & Shin, 1995; Hustedt & Beth, 1999; Hustedt & Beth, 2000; Persson et al, 2001). With the development of pulsed EPR methods such as double electron–electron resonance (DEER, also known as PELDOR) and double quantum coherence (DQC) the maximum distance measurable has been extended considerably.…”

PELDOR at S- and X-Band Frequencies and the Separation of Exchange Coupling from Dipolar Coupling

“…If it does not site-directed spin-labeling by nitroxides is a commonly used way to overcome this problem (8). Twofold spin-labeling makes it then possible to gather structural information about the labeled molecule by measuring the coupling energy E AB = hν AB between the two unpaired electrons with spins S A and S B (9). E AB is the eigenvalue of the electron-electron coupling Hamiltonian H AB [1], which is defined as the sum of the Hamiltonian H D [2] for the magnetic dipole-dipole coupling and the Hamiltonian H J [3] for the exchange coupling.…”

Site-directed Spin Labeling Studies on Nucleic Acid Structure and Dynamics

“…Hustrdt and Beth have categorized the majority of SDSL distance measurements into three cases (60,61). In the first case, called statically arranged spins, each nitroxide adopts a unique conformation with respect to the parent macromolecule, while rotational reorientation of the macromolecule can be treated as static on the EPR timescale (rotational correlation time is 1 μs or longer for X-band EPR).…”