Use of electron paramagnetic resonance to solve biochemical problems

Abstract

Electron paramagnetic resonance (EPR) spectroscopy is a very powerful biophysical tool that can provide valuable structural and dynamic information about a wide variety of biological systems. The intent of this review is to provide a general overview for biochemists and biological researchers of the most commonly used EPR methods and how these techniques can be used to answer important biological questions. The topics discussed could easily fill one or more textbooks; thus, we present a brief background on several important biological EPR techniques and an overview of several interesting studies that have successfully used EPR to solve pertinent biological problems. The review consists of the following sections: an introduction to EPR techniques, spin-labeling methods, and studies of naturally occurring organic radicals and EPR active transition metal systems that are presented as a series of case studies in which EPR spectroscopy has been used to greatly further our understanding of several important biological systems.

Figure 1

EPR transitions occur when the energy contained in the microwave photons matches the splitting between two electron spin states. In the simplest system, this splitting as a function of the magnetic field is gβ_eB₀.

Figure 2

Structure of MTSL and the resulting side chain produced by reaction with the cysteine residue of the protein. The _{χ1, χ2, χ3, χ4} and _χ5 represent the locations of five rotations about the chemical bonds between α-carbon backbone of the protein and the pyrroline ring of the attached MTSL.

Figure 3

Representation of TonB-dependent iron transporter: (A) X-ray crystal structure of Escherichia coli ferric citrate transporter FecA (PDB ID: 1KMO) (the N-terminal transcriptional signaling motif was not resolved in FecA), and (B) Solution NMR structure of the N-terminal transcriptional signaling motif of FecA (PDB ID: 1ZZV). Figures were prepared using visual molecular dynamics (VMD) software.

Figure 4

Schematic of the Vimentin molecular structure. Panel (A) shows a representation of the protein domains of Vimentin. At the amino terminus is the head domain, leading into rod domain 1. The black box between rod 1B and rod 2B represents Linker 1-2. The dark gray box between rod 1B and rod 2B represents the parallel helices structure of rod 2A/Linker 2. Panel (B) shows the amino acid sequence of the tail domain, beginning with Y. The HTM and β (beta) sites are boxed and labeled. Prolines are in bold; phosphorylation sites are indicated by asterisks. (Adapted from ref. with permission)

Figure 5

Representation of the NMR structure of KCNE1 membrane protein in LMPG micelles (PDB ID: 2K21). Figure was prepared using VMD software.

Figure 6

Representation of X-ray crystal structure of wild-type lactose permease protein (PDB ID: 2V8N). Figure was prepared using VMD software.

Figure 7

Representation of X-ray crystal structure of the motor domain of Dictyostelium discoideum myosin II (PDB ID: 1FMV). Figure was prepared using VMD software.

Figure 8

Three-pulse ESEEM experimental data with a τ=200ns of the i+2 and the i+3 ²H-labeled Leu for AchR M2δ helical peptide in lipid bilayer and ubiquitin β-sheet peptide in solution. (A) Time domain, (B) Frequency domain. The inset structural pictures show the location of spin labels and ²H-labeled Leu on AchR M2δ helical peptide and ubiquitin β-sheet peptide. (Adapted from ref. with permission)

Figure 9

DEER data used to identify the curvature helicity of the transmembrane region of Amyloid Precursor Protein (C99) in POPC/POPG vesicle system. (A) Topological illustration of C99 with respect to a lipid bilayer. (B) X-band DEER time domain data at 80 K for C99 that was spin-labeled at the ends of its TMD (at sites 700 and 723). Data are shown for WT C99 and also C99 that was additionally subjected to Gly-to-Leu mutations at G708 and G709. (C) Distance distributions between the spin labels measured for the corresponding time domain data. The error associated with each average distance relates to the uncertainty of the average. (Adapted from ref. with permission)

Figure 10

Secondary structure model of Na⁺/Proline Transporter PutP of E. coli. Putative TMs are represented as rectangles and numbered with Roman numerals; loops are numbered with Arabic numerals starting from the N-terminus. (Adapted from ref. with permission)

Figure 11

EPR spectra and corresponding simulations at three fields/frequencies of the radical intermediate in the Phycocyanobilin–Ferredoxin Oxidoreductase system. As the frequency increases, what is an isotropic signal at X-band eventually becomes much better resolved allowing for the determination of all three g values. Reproduced with permission from reference 119.

Figure 12

These data show the single crystal rotation of the H₂O₂-induced tyrosine radical intermediate in ribonucleotide reductase. From the results, the researchers were able to determine the orientation of the tyrosine sidechain within the crystal, which is slightly rotated from X-ray diffraction structure of the resting state. Reproduced with permission from reference 127.

Figure 13

X-ray crystal structure of metalloenzyme core of the oxygen evolving complex (OEC) (PDB ID: 3ARC). Atoms are shown in the following colors: Mn, purple; Ca, yellow; O, red. The figure was prepared using VMD.

Figure 14

Shown in this figure are the ⁵⁵Mn ESE ENDOR data collected by the Britt laboratory and the corresponding simulations using the hyperfine parameters from various predictions based on the multiline signal. The utility of using combined EPR methods is apparent as each of these sets of parameters can adequately simulate the multiline EPR signal in the S₂ state of the OEC, only the Britt values can be used to adequately simulate the ⁵⁵Mn ESE ENDOR spectrum. Reproduced with permission from reference 147.

Figure 15

X-ray crystal structure of FeMo cofactor of Nitrogenase (PDB ID: 3U7Q). Atoms are shown in the following colors: Mo, magenta; Fe, silver; S, yellow; Interstitial light atom, black. The figure was prepared using VMD.

Figure 16

¹⁵N ReMims ENDOR spectra of a trapped intermediate state in the Nitrogenase enzyme with ¹⁵N₂H₄, ¹⁵N₂H₂ or ¹⁵N=N-CH₃ (MD). The data show a single intermediate common to each of the added substrates. Adapted with permission from reference 167.