Vol 49, No 11 (2018)

Electron paramagnetic resonance (EPR) spectroscopy of spin-labeled lipids in complex with the sphingolipid activator proteins, GM2AP and SapB, was utilized to characterize the hydrophobic binding pocket of these lipid transfer proteins. Specifically, the EPR line shapes reveal that the mobility of the labeled lipids within the binding pockets of the transfer proteins are more restricted than when in a lipid bilayer environment and that lipids in GM2AP are slightly more restricted than in SapB. EPR accessibility based relaxation measurements show that the relative ratios of oxygen and water accessibility to sites along the acyl chains in lipids in complex with GM2AP are similar to the profiles obtained for a lipid bilayer albeit with lowered values. The results for SapB are quite different, with the oxygen profile mimicking a lipid bilayer, but there is a higher degree of water accessibility to the acyl chains in the SapB complex, likely because of the location of the lipid at the dimer interface in SapB coupled to dynamics of the dimer.

Heterogeneity of photooxidation processes in plaques made of commodity polymers, high-density polyethylene, and cyclic olefin copolymer poly(ethylene-co-norbornene) (Topas^® 8007) stabilized with various hindered amine stabilizers; some of them in combination with UV stabilizers exposed to accelerated weathering were studied using electron spin resonance imaging (ESRI) and three independent microscopic-scale methods: scanning electron microscopy, infrared microscopy, and microhardness testing. Concentration profiles of nitroxides mapping photooxidation process inside polymer plaques along the direction of incident radiation (perpendicular to the irradiated surface of the plaques) were determined by ESRI technique in dependence on the duration of the accelerated photooxidation. ESRI data were complemented with profiles of oxidation products, crystallinity, and microhardness measured inside the plaques along the same direction. Selection of stabilizers in the study was completed with α-tocopherol that is considered as the most active component of excellent processing stabilizer vitamin E.

S100A12 or Calgranulin C is a homodimeric antimicrobial protein of the S100 family of EF-hand calcium-modulated proteins. S100A12 is involved in many diseases such as inflammation, tumor invasion, cancer and neurological disorders such as Alzheimer’s disease. The binding of transition metal ions to the protein is important as the sequestering of the metal ion induces conformational changes in the protein, inhibiting the growth of various pathogenic microorganisms. In this work, we probe the Cu²⁺ binding properties of Calgranulin C. We demonstrate that the two Cu²⁺ binding sites in Calgranulin C show different coordination environments in solution. Continuous wave-electron spin resonance (CW-ESR) spectra of Cu²⁺-bound protein clearly show two distinct components at higher Cu²⁺:protein ratios, which is indicative of the two different binding environments for the Cu²⁺ ions. The g_|| and A_|| values are also different for the two components, indicating that the number of directly coordinated nitrogen in each site differs. Furthermore, we perform CW-ESR titrations to obtain the binding affinity of the Ca²⁺-loaded protein to Cu²⁺ ions. We observe a positive cooperativity in binding of the two Cu²⁺ ions. To further probe the Cu²⁺ coordination, we also perform electron spin echo envelope modulation (ESEEM) experiment. We perform ESEEM at two different fields where one Cu²⁺ binding site dominates the other. At both sites we see distinct signatures of Cu²⁺–histidine coordination. However, we clearly see that the ESEEM spectra corresponding to the two Cu²⁺ binding sites are significantly different. There is clear change in the intensity of the double quantum peak with respect to the nuclear quadrupole interaction peak at the two different fields. Furthermore, ESEEM along with hyperfine sublevel correlation show that only one of the two Cu²⁺ binding sites has backbone coordination, confirming our previous observation. Finally, we perform double electron–electron resonance spectroscopy to probe if the difference in binding environment is due to the Cu²⁺ binding to different sites in the protein. We obtain a distance distribution with a sharp peak at ~ 3 nm and a broad peak at ~ 4 nm. The shorter distance agrees with the Cu²⁺–Cu²⁺ distance expected for a dimer from the crystal structure. The longer distance is consistent with the Cu²⁺–Cu²⁺ distance when oligomerization occurs.