Pulsed EPR DEER structural studies of membrane proteins in a lipid bilayer have often been hindered by difficulties in extracting accurate distances when compared to globular proteins. In this study, we employed a combination of three recently developed methodologies: 1) bi-functional spin labels (BSL), 2) SMA-Lipodisq nanoparticles, and 3) Q-band pulsed EPR measurements to obtain improved signal sensitivity, increased transverse relaxation time, and more accurate and precise distances in DEER measurements on the integral membrane protein KCNE1. The KCNE1 EPR data indicated ~2 fold increase in the transverse relaxation time for the SMA-Lipodisq nanoparticles when compared to proteoliposomes, and narrower distance distributions for the BSL when compared to the standard MTSL. The certainty of information content in DEER data obtained for KCNE1 in SMA-Lipodisq nanoparticles is comparable to that in micelles. The combination of techniques will enable researchers to potentially obtain more precise distances in cases where the traditional spin labels and membrane systems yield imprecise distance distributions.

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An ESEEM (electron spin−echo envelope modulation) spectroscopic study employing a series of 2H-labeled alcohols provides direct evidence that small alcohols (methanol and ethanol) ligate to the Mn cluster of the oxygen evolving complex (OEC) of Photosystem II in the S2-state of the Kok cycle. A numerical method for calculating the through-space hyperfine interactions for exchange-coupled tetranuclear Mn clusters is described. This method is used to calculate hyperfine interaction tensors for protons [deuterons] in the vicinity of two different arrangements of Mn ions in a tetranuclear cluster:  a symmetric cubane model and the EXAFS-based Berkeley “dimer-of-dimers” model. The Mn−H distances derived from the spectroscopically observed coupling constants for methanol and ethanol protons [deuterons] and interpreted with these cluster models are consistent with the direct ligation of these small alcohols to the OEC Mn cluster. Specifically, for methanol we can simulate the three-pulse ESEEM time domain pattern with three dipolar hyperfine interactions of 2.92, 1.33, and 1.15 MHz, corresponding to a range of maximal Mn−H distances in the models of 3.7−5.6 Å (dimer-of-dimers) and 3.6−4.9 Å (symmetric cubane). We also find evidence for limited access of n-propanol, but no evidence for 2-propanol or DMSO access. Implications for substrate accessibility to the OEC are discussed.

This paper reports on the development of a new structural biology technique for determining the membrane topology of an integral membrane protein inserted into magnetically aligned phospholipid bilayers (bicelles) using EPR spectroscopy. The nitroxide spin probe, 2,2,6,6-tetramethylpiperidine-1-oxyl-4-amino-4-carboxylic acid (TOAC) was attached to the pore-lining transmembrane domain (M2δ) of the nicotinic acetylcholine receptor (AChR) and incorporated into a bicelle. The corresponding EPR spectra revealed hyperfine splittings that were highly dependent on the macroscopic orientation of the bicelles with respect to the static magnetic field. The helical tilt of the peptide can be easily calculated using the hyperfine splittings gleaned from the orientational dependent EPR spectra. A helical tilt of 14° was calculated for the M2δ peptide with respect to the bilayer normal of the membrane, which agrees well with previous 15 N solid-state NMR studies. The helical tilt of the peptide was verified by simulating the corresponding EPR spectra using the standardized MOMD approach. This new method is advantageous because: (1) bicelle samples are easy to prepare, (2) the helical tilt can be directly calculated from the orientational-dependent hyperfine splitting in the EPR spectra, and (3) EPR spectroscopy is approximately 1000 fold more sensitive than 15 N solid-state NMR spectroscopy; thus, the helical tilt of an integral membrane peptide can be determined with only 100 μg of peptide. The helical tilt can be determined more accurately by placing TOAC spin labels at several positions with this technique.

EPR spectroscopy is a very powerful biophysical tool that can provide valuable structural and dynamic information on a wide variety of biological systems. The intent of this review is to provide a general overview for biochemists and biological researchers on 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 which 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.

Four new metal-organic frameworks (MOFs) containing chiral channels have been synthesized using an achiral, triazine-based trigonal-planar ligand, 4,4',4' '-s-triazine-2,4,6-triyltribenzoate (TATB), and an hourglass secondary building unit (SBU): Zn3(TATB)2(H2O)2.4DMF.6H2O (1); Cd3(TATB)2(H2O)2.7DMA.10H2O (2); [H2N(CH3)2][Zn3(TATB)2(HCOO)].HN(CH3)2.3DMF.3H2O (3); [H2N(CH3)2][Cd3(TATB)2(CH3COO)].HN(CH3)2.3DMA.4H2O (4). MOFs 1 and 2 are isostructural and possess (10,3)-a nets containing large chiral channels of 20.93 and 21.23 A, respectively, but are thermally unstable due to the easy removal of coordinated water molecules on the SBU. Replacement of these water molecules by formate or acetate generated in situ leads to 3 and 4, respectively. Formate or acetate links SBUs to form infinite helical chains bridged by TATB to create three-dimensional anionic networks, in which one of the two oxygen atoms of the formate or acetate is uncoordinated and points into the void of the channels. This novel SBU-stabilization and channel-functionalization strategy may have general implications in the preparation of new MOFs. Thermogravimetric analysis (TGA) shows that solvent-free 3' is thermally stable to 410 degrees C, while TGA studies on samples vapor-diffused with water, methanol, and chloroform show reversible adsorption. MOF 3 also has permanent porosity with a large Langmuir surface area of 1558 m2/g. All complexes exhibit similar strong luminescence with a lambdamax of approximately 423 nm upon excitation at 268.5 nm.

Characterization of membrane proteins is challenging due to the difficulty in mimicking the native lipid bilayer with properly folded and functional membrane proteins. Recently, styrene-maleic acid (StMA) copolymers have been shown to facilitate the formation of disc-like lipid bilayer mimetics that maintain the structural and dynamic integrity of membrane proteins. Here we report the controlled synthesis and characterization of StMA containing block copolymers. StMA polymers with different compositions and molecular weights were synthesized and characterized by size exclusion chromatography (SEC). These polymers act as macromolecular surfactants for 1-Palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC)/1-palmitoyl-2-oleoyl-sn-glycero-3-phospho-(1'-rac-glycerol (POPG) lipids, forming disc like structures of the lipids with the polymer wrapping around the hydrophobic lipid edge. A combination of dynamic light scattering (DLS), solid-state nuclear magnetic resonance (SSNMR) spectroscopy, and transmission electron microscopy (TEM) was used to characterize the size of the nanoparticles created using these StMA polymers. At a weight ratio of 1.25:1 StMA to lipid, the nanoparticle size created is 28+1nm for a 2:1 ratio, 10+1nm for a 3:1 StMA ratio and 32+1nm for a 4:1 StMA ratio independent of the molecular weight of the polymer. Due to the polymer acting as a surfactant that forms disc like nanoparticles, we term these StMA based block copolymers "RAFT SMALPs". RAFT SMALPs show promise as a new membrane mimetic with different nanoscale sizes, which can be used for a wide variety of biophysical studies of membrane proteins.

The molecular oxygen in our atmosphere is a product of a water-splitting reaction that occurs in the oxygenevolving complex of photosystem II of oxygenic photosynthesis. The catalytic core of the oxygen-evolving complex Is an ensemble of four manganese atoms arranged in a cluster of undetermined structure. The pulsed electron paramagnetic resonance (EPR) technique of electron spin-echo envelope modulation (ESEEM) can be used to measure nuclear spin transitions of nuclei magnetically coupled to paramagnetic metal centers of enzymes. We report the results of ESEEM experiments on the cyanobacterium Synechocystis PCC 6803 selectively labeled with 15N at the two nitrogen sites of the imidazole side chain of histidine residues. The experiments demonstrate that histidine is bound to manganese in the oxygen-evolving complex.Photosynthetic oxygen evolution is a cyclic process involving five redox states, So through S4, where the subscript refers to the number of oxidizing equivalents that have accumulated on the oxygen-evolving complex (OEC) (1). These accumulate sequentially with each light-driven charge separation of the photosystem II (PSII) reaction center. Upon attaining the S4 state an oxygen molecule is released, and the OEC returns to the So state. The catalytic core of the OEC consists of a tetranuclear manganese cluster of undetermined structure. Electron paramagnetic resonance (EPR) signals arising from the manganese cluster of the OEC poised in the S2 state have been observed in the g = 2 and g = 4 regions of the EPR spectrum (2-5). The first electron spin-echo envelope modulation (ESEEM) study of the g = 2 "multiline" signal, using PSII membranes isolated from spinach, revealed a broad structured peak centered near 5 MHz (6). This peak was shown to arise from 14N by ESEEM experiments on PSII particles isolated from a thermophilic cyanobacterium (Synechococcus sp.) grown alternatively with MATERIALS AND METHODSIsolation of a Histidine-Tolerant Strain. Labarre et al. (15) described the isolation of a histidine-tolerant mutant of Synechocystis PCC 6803 in which the transport of this normally toxic amino acid was impaired. As our application required incorporation of labeled histidine into cellular protein, we needed to both maintain histidine transport in a histidine-tolerant organism and suppress endogenous biosynthesis. The latter could be accomplished either through inactivation of genes involved in the biosynthetic pathway or through feedback inhibition of the pathway by the exogenous amino acid. A spontaneous histidine-tolerant strain showing feedback inhibition of the histidine biosynthetic pathway was obtained by growing wild-type cells photoautotrophically in Preparation of PSII Oxygen-Evolving Core Complexes.The histidine-tolerant strain was grown photoautotrophically in two 10-liter batches of BG-11 medium bubbled with 5% CO2 in air and containing 240 uM DL-histidine. In one batch, the histidine contained only natural-abundance 14N. In the other batch, both of the imidazole nitrogens were...