Mouse Pxt1 expression is regulated by Mir6996 miRNA.

Mouse Pxt1 gene is expressed exclusively in male germ cells and encodes for a small, cell death inducing protein. However, upon PXT1 interaction with BAG6, cell death is prevented. In transiently transfected cell lines the PXT1 expression triggered massive cell death, thus we ask the question whether the interaction of PXT1 and BAG6 is the only mechanism preventing normal, developing male germ cells from being killed by PXT1. The Pxt1 gene contains a long 3'UTR thus we have hypothesized that Pxt1 can be regulated by miRNA. We have applied Pxt1 knockout and used Pxt1 transgenic mice that overexpressed this gene to shed more light on Pxt1 regulation. Using the ELISA assay we have demonstrated that PXT1 protein is expressed in adult mouse testis, though at low abundance. The application of dual-Glo luciferase assay and the 3'UTR cloned into p-MIR-Glo plasmid showed that Pxt1 is regulated by miRNA. Combining the use of mirDB and the site-directed mutagenesis further demonstrated that Pxt1 translation is suppressed by Mir6996-3p. Considering previous reports and our current results we propose a model for Pxt1 regulation in the mouse male germ cells.

Local water sensing: water exchange in bacterial photosynthetic reaction centers embedded in a trehalose glass studied using multiresonance EPR.

Using isotope labeled water (D2O and H217O) and pulsed W-band (94 GHz) high-field multiresonance EPR spectroscopies, such as ELDOR-detected NMR and ENDOR, the biologically important question of detection and quantification of local water in proteins is addressed. A bacterial reaction center (bRC) from Rhodobacter sphaeroides R26 embedded into a trehalose glass matrix is used as a model system. The bRC hosts the two native radical cofactor ions (primary electron donor) and (primary electron acceptor) as well as an artificial nitroxide spin label site-specifically attached to the surface of the H-protein domain. The three paramagnetic reporter groups have distinctly different local environments. They serve as local probes to detect water molecules via magnetic interactions (electron-nuclear hyperfine and quadrupole) with either deuterons or 17O nuclei. bRCs were equilibrated in an atmosphere of different relative humidities allowing us to control precisely the hydration levels of the protein. We show that by using oxygen-17 labeled water quantitative conclusions can be made in contrast to using D2O which suffers from proton-deuterium exchange processes in the protein. From the experiments we also conclude that dry trehalose operates as an anhydrobiotic protein stabilizer in line with the "anchorage hypothesis" of bio-protection. It predicts selective changes in the first solvation shell of the protein upon trehalose-matrix dehydration with subsequent changes in the hydrogen-bonding network. Changes in hydrogen-bonding patterns usually have an impact on the global function of a biological system.

ELDOR-detected NMR: A general and robust method for electron-nuclear hyperfine spectroscopy?

ELDOR-detected NMR (EDNMR) performed at higher magnetic fields is becoming an increasingly popular alternative to conventional ENDOR for the characterization of electron-nuclear hyperfine interactions owing to its enhanced sensitivity. However there are two key problems that limit its widespread adoption, with factors controlling: (i) lineshape distortions and; (ii) overall spectral resolution, still largely understood only at a qualitative level. Indeed highly anisotropic (dipolar) coupled species are particularly problematic in the EDNMR experiment. Nor is it clear as to whether line intensities measured in EDNMR can provide quantitative information. Here we describe how all these problems can be overcome for a nitroxide radical as model system. We introduce a simulation procedure/protocol for the simulation of EDNMR line-shapes collected over a range of high turning angle (HTA) pulse lengths. It is shown that spectral line-shapes can be robustly reproduced and that the intensities of spectral lines and the spin nutation behavior can be quantitatively assessed. This broadens the scope of the EDNMR experiment as a generally applicable, quantitative double resonance method.

Computing distance distributions from dipolar evolution data with overtones: RIDME spectroscopy with Gd(iii)-based spin labels.

Extraction of distance distributions between high-spin paramagnetic centers from relaxation induced dipolar modulation enhancement (RIDME) data is affected by the presence of overtones of dipolar frequencies. As previously proposed, we account for these overtones by using a modified kernel function in Tikhonov regularization analysis. This paper analyzes the performance of such an approach on a series of model compounds with the Gd(iii)-PyMTA complex serving as paramagnetic high-spin label. We describe the calibration of the overtone coefficients for the RIDME kernel, demonstrate the accuracy of distance distributions obtained with this approach, and show that for our series of Gd-rulers RIDME technique provides more accurate distance distributions than Gd(iii)-Gd(iii) double electron-electron resonance (DEER). The analysis of RIDME data including harmonic overtones can be performed using the MATLAB-based program OvertoneAnalysis, which is available as open-source software from the web page of ETH Zurich. This approach opens a perspective for the routine use of the RIDME technique with high-spin labels in structural biology and structural studies of other soft matter.

Pulse Double-Resonance EPR Techniques for the Study of Metallobiomolecules.

Electron paramagnetic resonance (EPR) spectroscopy exploits an intrinsic property of matter, namely the electron spin and its related magnetic moment. This can be oriented in a magnetic field and thus, in the classical limit, acts like a little bar magnet. Its moment will align either parallel or antiparallel to the field, giving rise to different energies (termed Zeeman splitting). Transitions between these two quantized states can be driven by incident microwave frequency radiation, analogous to NMR experiments, where radiofrequency radiation is used. However, the electron Zeeman interaction alone provides only limited information. Instead, much of the usefulness of EPR is derived from the fact that the electron spin also interacts with its local magnetic environment and thus can be used to probe structure via detection of nearby spins, e.g., NMR-active magnetic nuclei and/or other electron spin(s). The latter is exploited in spin labeling techniques, an exciting new area in the development of noncrystallographic protein structure determination. Although these interactions are often smaller than the linewidth of the EPR experiment, sophisticated pulse EPR methods allow their detection. A number of such techniques are well established today and can be broadly described as double-resonance methods, in which the electron spin is used as a reporter. Below we give a brief description of pulse EPR methods, particularly their implementation at higher magnetic fields, and how to best exploit them for studying metallobiomolecules.

Changes in the Microenvironment of Nitroxide Radicals around the Glass Transition Temperature.

For structural characterization by pulsed EPR methods, spin-labeled macromolecules are routinely studied at cryogenic temperatures. The equilibration of the conformational ensemble during shock-freezing occurs to a good approximation at the glass transition temperature (Tg). In this work, we used X-band power saturation continuous wave (cw) EPR to obtain information on the glass transition temperatures in the microenvironment of nitroxide radicals in solvents or bound to different sites in proteins. The temperature dependence of the saturation curve of nitroxide probes in pure glycerol or ortho-terphenyl showed detectable transitions at the respective Tg values, with the latter solvent characterized by a sharper change of the saturation properties, according to its higher fragility. In contrast, nitroxide probes in a glycerol/water mixture showed a discontinuity in the saturation properties close to the expected glass transition temperature, which made the determination of Tg complicated. Low-temperature W-band cw EPR and W-band ELDOR-detected NMR experiments demonstrated that the discontinuity is due to local rearrangements of H-bonds between water molecules and the nitroxide reporter group. The change in the network of H-bonds formed between the nitroxide and water molecules that occurs around Tg was found to be site-dependent in spin-labeled proteins. This effect can therefore be modulated by neighboring residues with different steric hindrances and/or charge distributions and possibly by the glycerol enrichment on protein surfaces. In conclusion, if the thermal history of the sample is carefully reproduced, the nitroxide probe is extremely sensitive in reporting site-specific changes in the H-bonding to water molecules close to Tg and local glass transition temperatures in spin-labeled macromolecules.

High-field ELDOR-detected NMR study of a nitroxide radical in disordered solids: towards characterization of heterogeneity of microenvironments in spin-labeled systems.

The combination of high-field EPR with site-directed spin-labeling (SDSL) techniques employing nitroxide radicals has turned out to be particularly powerful in probing the polarity and proticity characteristics of protein/matrix systems. This information is concluded from the principal components of the nitroxide Zeeman (g), nitrogen hyperfine (A) and quadrupole (P) tensors of the spin labels attached to specific sites. Recent multi-frequency high-field EPR studies underlined the complexity of the problem to treat the nitroxide microenvironment in proteins adequately due to inherent heterogeneities which result in several principal x-components of the nitroxide g-tensor. Concomitant, but distinctly different nitrogen hyperfine components could, however, not be determined from high-field cw EPR experiments owing to the large intrinsic EPR linewidth in fully protonated guest/host systems. It is shown in this work that, using the W-band (95GHz) ELDOR- (electron-electron double resonance) detected NMR (EDNMR) method, different principal nitrogen hyperfine, Azz, and quadrupole, Pzz, tensor values of a nitroxide radical in glassy 2-propanol matrix can be measured with high accuracy. They belong to nitroxides with different hydrogen-bond situations. The satisfactory resolution and superior sensitivity of EDNMR as compared to the standard ENDOR (electron-nuclear double resonance) method are demonstrated.

RIDME Spectroscopy with Gd(III) Centers.

The relaxation induced dipolar modulation enhancement (RIDME) technique is applied at W-band microwave frequencies around 94 GHz to a pair of Gd(III) complexes that are connected by a rodlike spacer, and the extraction of the interspin distance distribution is discussed. A dipolar pattern derived from RIDME experimental data is a superposition of Pake-like dipolar patterns corresponding to the fundamental dipolar interaction and higher harmonics thereof. Intriguingly, the relative weights of the stretched patterns do not depend significantly on mixing time. As much larger modulation depths can be achieved than in double electron-electron resonance distance measurements at the same frequency, Gd(III)-Gd(III) RIDME may become attractive for structural characterization of biomacromolecules and biomolecular complexes.

Preparation of cysteine-34-nitroxide spin labeled human α1-microglobulin.

α(1)-Microglobulin (α(1)m) is a protein of yet unresolved function occurring in blood plasma and urine. It consists of a lipocaline type of fold with two cysteine residues forming a disulfide bridge and the third cysteine-34 remaining a free, somewhat reactive thiol. A number of investigations point to an interaction with heme and we have recently reported, that heme binding triggers the formation of a stable α(1)m trimer upon modification of cysteine-34 with 2-iodoacetamide, i.e., [α(1)m(heme)(2)](3) [J.F. Siebel, R.L. Kosinsky, B. Åkerström, M. Knipp, Insertion of heme b into the structure of the Cys34-carbamidomethylated human lipocalin α(1)-microglobulin-formation of a [(heme)(2)(α(1)-microglobulin)](3) complex, ChemBioChem 13 (2012) 879-887]. For further structural and functional investigations, an improved purification protocol for α(1)m was sought, in particular yielding an untagged amino acid sequence. The method reported herein improves the speed and the yield of the protein production even when an expression plasmid without tag was applied. Furthermore, for the purpose of future structural studies using electron paramagnetic resonance (EPR) techniques, in accordance to the modification with 2-iodoacetamide (α(1)m(AM)), the protein was modified with 3-(2-iodoacetamido)-2,2,5,5-tetramethyl-1-pyrrolidinyloxy (3-(2-iodoacetamido)-PROXYL) yielding the nitroxide spin labeled α(1)m(N-O). The extinction coefficient of the protein was calibrated using magnetic circular dichroism (MCD) spectroscopy of tryptophan (ε(280nm)=40,625M(-1)cm(-1)). The parallel quantification by absorbance spectroscopy (protein) and cw-EPR spectroscopy (radical spin) determined the degree of spin labeling to 90%. Characterization of the protein by circular dichroism (CD) spectroscopy and matrix assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) upon tryptic digestion further demonstrated the similar fold of α(1)m(AM) and α(1)m(N-O), but also established the modification of cystein-34 as well as the formation of the cysteine-72-cysteine-169 disulfide bond.