The biological function of protein, DNA, and RNA molecules often depends on relative movements of domains with dimensions of a few nanometers. This length scale can be accessed by distance measurements between spin labels if pulsed electron paramagnetic resonance (EPR) techniques such as electron-electron double resonance (ELDOR) and double-quantum EPR are used. The approach does not require crystalline samples and is well suited to biomacromolecules with an intrinsic flexibility as distributions of distances can be measured. Furthermore, oligomerization or complexation of biomacromolecules can also be studied, even if it is incomplete. The sensitivity of the technique and the reliability of the measured distance distribution depend on careful optimization of the experimental conditions and procedures for data analysis. Interpretation of spin-to-spin distance distributions in terms of the structure of the biomacromolecules furthermore requires a model for the conformational distribution of the spin labels.

Studies of structure and dynamics of proteins using site-directed spin labelling rely on explicit modelling of spin label conformations. The large computational effort associated with such modelling with molecular dynamics (MD) simulations can be avoided by a rotamer library approach based on a coarse-grained representation of the conformational space of the spin label. We show here, that libraries of about 200 rotamers, obtained by iterative projection of a long MD trajectory of the free spin label onto a set of canonical dihedral angles, provide a representation of the underlying trajectory adequate for EPR distance measurements. Rotamer analysis was performed on selected Xray structures of spin labelled T4 lysozyme mutants to characterize the spin label rotamer ensemble on a single protein site. Furthermore, predictions based on the rotamer library approach are shown to be in nearly quantitative agreement with electron paramagnetic resonance (EPR) distance data on Na + /H + antiporter NhaA and on the light-harvesting complex LHCII whose structures are known from independent cryo electron microscopy and X-ray studies, respectively. Suggestions for the selection of labelling sites in proteins are given, limitations of the approach discussed, and requirements for further development are outlined.3

Measurement of distances with the Double Electron-Electron Resonance (DEER) experiment at X-band frequencies using a pair of nitroxides as spin labels is a popular biophysical tool for studying function-related conformational dynamics of proteins. The technique is intrinsically highly precise and can potentially access the range from 1.5 to 6-10 nm. However, DEER performance drops strongly when relaxation rates of the nitroxide spin labels are high and available material quantities are low, which is usually the case for membrane proteins reconstituted into liposomes. This leads to elevated noise levels, very long measurement times, reduced precision, and a decrease of the longest accessible distances. Here we quantify the performance improvement that can be achieved at Q-band frequencies (34.5 GHz) using a high-power spectrometer. More than an order of magnitude gain in sensitivity is obtained with a homebuilt setup equipped with a 150 W TWT amplifier by using oversized samples. The broadband excitation enabled by the high power ensures that orientation selection can be suppressed in most cases, which facilitates extraction of distance distributions. By varying pulse lengths, Q-band DEER can be switched between orientationally non-selective and selective regimes. Because of suppression of nuclear modulations from matrix protons and deuterons, analysis of the Q-band data is greatly simplified, particularly in cases of very small DEER modulation depth due to low binding affinity between proteins forming a complex or low labelling efficiency. Finally, we demonstrate that a commercial Q-band spectrometer can be readily adjusted to the high-power operation.

High-density lipoprotein (HDL) particles are cholesterol and lipid transport containers. Mature HDL particles destined for the liver develop through the formation of intermediate discoidal HDL particles, which are the primary acceptors for cholesterol. Here we present the three-dimensional structure of reconstituted discoidal HDL (rdHDL) particles, using a shortened construct of human apolipoprotein A-I, determined from a combination of nuclear magnetic resonance (NMR), electron paramagnetic resonance (EPR) and transmission electron microscopy (TEM) data. The rdHDL particles feature a protein double belt surrounding a lipid bilayer patch in an antiparallel fashion. The integrity of this structure is maintained by up to 28 salt bridges and a zipper-like pattern of cation-π interactions between helices 4 and 6. To accommodate a hydrophobic interior, a gross 'right-to-right' rotation of the helices after lipidation is necessary. The structure reflects the complexity required for a shuttling container to hold a fluid lipid or cholesterol interior at a protein:lipid ratio of 1:50.

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