We describe a two-dimensional solid-state NMR technique to investigate membrane protein topology under magic-angle spinning conditions. The experiment detects the rate of (1)H spin diffusion from the mobile lipids to the rigid protein. While spin diffusion within the rigid protein is fast, magnetization transfer in the mobile lipids is an inefficient and slow process. Qualitative analysis of (1)H spin-diffusion build-up curves from the lipid chain-end methyl groups to the protein allows the identification of membrane-embedded domains in the protein. Numerical simulations of spin-diffusion build-up curves yield the approximate insertion depth of protein segments in the membrane. The experiment is demonstrated on the selectively (13)C labeled colicin Ia channel domain, known to have a membrane-embedded domain, and on DNA/cationic lipid complexes where the DNA rods are bound to the membrane surface. The experiment is designed for X-nucleus detection, which could be (13)C or (15)N in the protein and (31)P for the DNA. Finally, we show that a qualitative distinction between membrane proteins with and without a membrane-embedded domain can be made even by using an unlabeled protein, by detection of lipid signals. This spin-diffusion experiment is simple to perform and requires no oriented bilayer preparations and only standard NMR hardware.

Genetically-encoded protein photosensors, including the LOV (light, oxygen, voltage) domain, are promising tools for engineering optical control of cellular behavior. We are only beginning to understand how to couple these light detectors to effectors of choice. We report a method that increases the dynamic range of an artificial photoswitch based on the LOV2 domain of A. sativa phototropin1 (AsLOV2). This approach can potentially be used to improve many AsLOV2-based photoswitches.

Protein photosensors provide versatile tools to study ligand-regulated allostery and signaling. Fundamental to these processes is the amount of energy that can be provided by a photosensor to control downstream signaling events. Such regulation is exemplified by the phototropins, plant serine/threonine kinases that are activated by blue light via conserved LOV (Light, Oxygen and Voltage) domains. The core photosensor of oat phototropin 1 is a LOV domain that interacts in lightdependent fashion with an adjacent α-helix (Jα) to control kinase activity. We used solution NMR measurements to quantify the free energy of the LOV domain:Jα helix binding equilibrium in the dark and lit states. These data indicate that light shifts this equilibrium by ~3.8 kcal mol −1 , quantifying the energy available through LOV-Jα for light-driven allosteric regulation. This study provides insight into the energetics of light sensing by phototropins and benchmark values for engineering photoswitchable systems based on the LOV-Jα interaction. Keywordsphototropin; free energy; conformational dynamics; NMR; relaxation dispersion Information transfer in biological signaling pathways often takes the form of stimulus-induced changes in protein structure that consequently alter a functional output. Many signaling proteins are composed of linked arrays of modular domains, which cooperatively function to control activity 1,2 . In such proteins, sensor domains receive inputs related to changes in the local environment, e.g. the binding of a metabolite or other protein, or the alteration of a bound cofactor by a change in redox state. These events change the interactions of the sensors with other domains, which subsequently transmit the signal downstream through changes in their activities. A critical parameter in the construction of such sensors is the magnitude of inputinduced changes in the energetics of the sensor domain and its interactions with downstream effectors. It is this change in energy, and the corresponding change in interaction equilibria, that determines the dynamic range of the sensor along with several other fundamental signaling properties.Several examples of this principle are provided by photosensory proteins, which have evolved to sense and respond to light across the UV/visible spectrum at a wide range of intensities 3 . These sensory processes are typically achieved through protein domains that bind lightabsorbing chromophores and convert photon energy into structural, dynamic and functional changes. One class of these proteins are phototropins, a group of blue light-activated serine/

We demonstrate the use of Lee-Goldburg cross-polarization (LG-CP) NMR under fast magic-angle spinning (MAS) to investigate the amplitude and geometry of segmental motions in biomolecular and polymeric solids. Motional geometry information was previously available only from 2 H NMR, which, however, has limited site resolution and requires site-specific isotopic labeling. Using a 2D LG-CP technique, we resolve the 13 C-1 H or 15 N-1 H dipolar couplings according to the 13 C or 15 N isotropic chemical shift. Applications to systems undergoing 180°phenylene ring flips show spectral line shapes reflecting the geometry of the motion. Using this LG-CP technique, we measured the 13 C-1 H and 15 N-1 H dipolar couplings in the water-soluble and membrane-bound states of the colicin Ia channel domain. The backbone motions of the membrane-bound colicin scale both the CR-HR and N-H couplings similarly, thus ruling out rotation of the R-helices around their axes as a specific mechanism of motion. We also show that the sensitivity of the LG-CP spectra can be enhanced by the addition of a phase-inverted 1 H-13 C cross-polarization step, and the site resolution of the 15 N-1 H LG-CP spectra can be enhanced by 13 C indirect detection.

Electrical resistivity () and Seebeck-coefficient measurements (␣) are reported for NiS 2Ϫx Se x single crystals in the range 0рxр0.71. There is a general trend toward increasing metallicity with increasing x. In the range 0.38рxр0.51 a pronounced rise of with temperature (T) is observed where the antiferromagnetic insulating ͑or antiferromagnetic metallic͒ phase changes over to the paramagnetic insulating phase. The analysis of ␣ vs T curves suggests that in the low-temperature insulating state both holes and electrons participate in charge transport. It is emphasized that the many changes in electrical characteristics occur without significant alterations in the pyrite crystal structure, and that physical properties are greatly altered solely by adjustment of the anion sublattice while the cation sublattice remains intact. The results concerning electrical transport for the samples with 0.38рxр0.55 are interpreted qualitatively on the basis of an onset of the Hubbard splitting into subbands at the transition to the insulating ͑semiconducting͒ state, which takes place in the temperature interval ͑50-100 K͒ upon heating the samples. The metallic state close to the metalsemiconductor boundary is viewed as being an antiferromagnetic semimetal, with an anisotropic Slater gap.

Channel-forming colicins are bactericidal proteins that spontaneously insert into hydrophobic lipid bilayers. We have used magic-angle spinning solid-state nuclear magnetic resonance spectroscopy to examine the conformational differences between the water-soluble and the membrane-bound states of colicin Ia channel domain, and to study the effect of bound colicin on lipid bilayer structure and dynamics. We detected (13)C and (15)N isotropic chemical shift differences between the two forms of the protein, which indicate structural changes of the protein due to membrane binding. The Val C(alpha) signal, unambiguously assigned by double-quantum experiments, gave a 0.6 ppm downfield shift in the isotropic position and a 4 ppm reduction in the anisotropic chemical shift span after membrane binding. These suggest that the alpha-helices in the membrane-bound colicin adopt more ideal helical torsion angles as they spread onto the membrane. Colicin binding significantly reduced the lipid chain order, as manifested by (2)H quadrupolar couplings. These results are consistent with the model that colicin Ia channel domain forms an extended helical array at the membrane-water interface upon membrane binding.

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