Comparative evaluation of spin-label modeling methods for protein structural studies.

Site-directed spin-labeling electron paramagnetic resonance spectroscopy is a powerful technique for the investigation of protein structure and dynamics. Accurate spin-label modeling methods are essential to make full quantitative use of site-directed spin-labeling electron paramagnetic resonance data for protein modeling and model validation. Using a set of double electron-electron resonance data from seven different site pairs on maltodextrin/maltose-binding protein under two different conditions using five different spin labels, we compare the ability of two widely used spin-label modeling methods, based on accessible volume sampling and rotamer libraries, to predict experimental distance distributions. We present a spin-label modeling approach inspired by canonical side-chain modeling methods and compare modeling accuracy with the established methods.

Mechanism of Electron Spin Decoherence in a Partially Deuterated Glassy Matrix.

Long electron spin coherence lifetimes are essential for applications in quantum information science and electron paramagnetic resonance, for instance, for nanoscale distance measurements in biomolecular systems using double electron-electron resonance. We experimentally investigate the decoherence dynamics under the Hahn echo sequence of the organic radical d18-TEMPO in a variably deuterated frozen water:glycerol matrix. The coherence time (phase memory time) TM scales with proton concentration as [1H]-0.65. For selectively deuterated matrices, decoherence is accelerated in the presence of proton clustering, that is, substantial short-range density in the proton-proton radial distribution functions (<3 Å). Simulations using molecular dynamics and many-body spin quantum dynamics show excellent agreement with experiment and show that geminal proton pairs such as CH2 and OH2 groups are major decoherence drivers. This provides a predictive tool for designing molecular systems with long electron spin coherence times.

Quantitative Structure-Based Prediction of Electron Spin Decoherence in Organic Radicals.

The decoherence, or dephasing, of electron spins in paramagnetic molecules limits sensitivity and resolution in electron paramagnetic resonance spectroscopy, and it represents a challenge for utilizing paramagnetic molecules as qubit units in quantum information devices. For organic radicals in dilute frozen aqueous solution at cryogenic temperatures, electron spin decoherence is driven by neighboring nuclear spins. Here, we show that this nuclear-spin-driven decoherence can be quantitatively predicted from the molecular structure and solvation geometry of the radicals. We use a fully deterministic quantum model of the electron spin and up to 2000 neighboring protons with a static spin Hamiltonian that includes nucleus-nucleus couplings. We present experiments and simulations of two nitroxide radicals and one trityl radical, which have decoherence time scales of 4-5 µs below 60 K. We show that nuclei within 12 Å of the electron spin contribute to decoherence, with the strongest impact from protons 4-8 Å away.

FBXL5 Regulates IRP2 Stability in Iron Homeostasis via an Oxygen-Responsive [2Fe2S] Cluster.

Cellular iron homeostasis is dominated by FBXL5-mediated degradation of iron regulatory protein 2 (IRP2), which is dependent on both iron and oxygen. However, how the physical interaction between FBXL5 and IRP2 is regulated remains elusive. Here, we show that the C-terminal substrate-binding domain of FBXL5 harbors a [2Fe2S] cluster in the oxidized state. A cryoelectron microscopy (cryo-EM) structure of the IRP2-FBXL5-SKP1 complex reveals that the cluster organizes the FBXL5 C-terminal loop responsible for recruiting IRP2. Interestingly, IRP2 binding to FBXL5 hinges on the oxidized state of the [2Fe2S] cluster maintained by ambient oxygen, which could explain hypoxia-induced IRP2 stabilization. Steric incompatibility also allows FBXL5 to physically dislodge IRP2 from iron-responsive element RNA to facilitate its turnover. Taken together, our studies have identified an iron-sulfur cluster within FBXL5, which promotes IRP2 polyubiquitination and degradation in response to both iron and oxygen concentrations.

Biochemical and spectroscopic characterization of dinuclear Mn-sites in artificial four-helix bundle proteins.

To better understand metalloproteins with Mn-clusters, we have designed artificial four-helix bundles to have one, two, or three dinuclear metal centers able to bind Mn(II). Circular dichroism measurements showed that the Mn-proteins have substantial α-helix content, and analysis of electron paramagnetic resonance spectra is consistent with the designed number of bound Mn-clusters. The Mn-proteins were shown to catalyze the conversion of hydrogen peroxide into molecular oxygen. The loss of hydrogen peroxide was dependent upon the concentration of protein with bound Mn, with the proteins containing multiple Mn-clusters showing greater activity. Using an oxygen sensor, the oxygen concentration was found to increase with a rate up to 0.4µM/min, which was dependent upon the concentrations of hydrogen peroxide and the Mn-protein. In addition, the Mn-proteins were shown to serve as electron donors to bacterial reaction centers using optical spectroscopy. Similar binding of the Mn-proteins to reaction centers was observed with an average dissociation constant of 2.3µM. The Mn-proteins with three metal centers were more effective at this electron transfer reaction than the Mn-proteins with one or two metal centers. Thus, multiple Mn-clusters can be incorporated into four-helix bundles with the capability of performing catalysis and electron transfer to a natural protein.