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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 like inflammation, tumor invasion, cancer and neurological disorders like 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 Cu(II) binding properties of Calgranulin C. We demonstrate that the two Cu(II) binding sites in Calgranulin C show different coordination environments in solution. Electron spin resonance (ESR) spectra of Cu(II)-bound protein clearly show two distinct components at higher Cu(II):protein ratios, which is indicative of the two different binding environments for the Cu(II) ions. The g|| and A|| values are also different for the two components, indicating that the number of directly coordinated nitrogens in each site differs. Furthermore, we perform Continuous Wave (CW)-titrations to obtain the binding affinity of the Ca(II)-loaded protein to Cu2+ ions. We observe a positive cooperativity in binding of the two Cu(II) ions. In order 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 Cu(II) binding site dominates over the other. At both sites we see distinct signatures of Cu(II)-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 (DQ) peak with respect to the nuclear quadrupole interaction (NQI) peak at the two different fields. Furthermore, ESEEM along with Hyperfine Sublevel Correlation (HYSCORE) show that only one of the two Cu(II) binding sites has backbone coordination, confirming our previous observation. Finally, we perform Double Electron Electron Resonance (DEER) spectroscopy to probe if the difference in binding environment is due to the Cu(II) 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 Cu(II)-Cu(II) distance expected for a dimer from the crystal structure. The longer distance is consistent with the Cu(II)-Cu(II) distance when oligomerization occurs. 

Abstract Travertine deposits preserve an invaluable record of both ancient and modern fluid flow. The goal of this study is to reconstruct spatial and temporal patterns in travertine deposition associated with tectonic and climatic controls along the Lucero Uplift in New Mexico, USA. Uranium‐series ages of travertine deposits in the Lucero Uplift range from 0.94 ± 0.01 to 592 ± 110 ka, indicating that travertine formation has been episodically active since at least ∼600 ka. We find minimal evidence to attribute glacial and interglacial cycles to travertine formation in the Lucero Uplift. δ¹³C values in travertine deposits range from 2‰ to 9‰ (Vienna Pee Dee Belemnite), δ¹⁸O values range from 21‰ to 25‰ (Vienna Standard Mean Ocean Water). Positive correlation between travertine δ¹³C and δ¹⁸O values indicate travertine formation is closely associated with various degrees of CO₂degassing.⁸⁷Sr/⁸⁶Sr values in travertine deposits range from 0.714 to 0.717 and (²³⁴U/²³⁸U)_ivalues exhibit a remarkably wide range from 3.6 to 9.3, indicative of fluid‐rock interaction during deep crustal circulation in more radiogenic basement rocks. Reconstructed δ¹³C, δ¹⁸O, and (²³⁴U/²³⁸U)_ivalues in the inferred deep fluid sources showed systematic variations with travertine formation ages, while⁸⁷Sr/⁸⁶Sr values remain relatively constant. Based on dating of undeformed travertine deposits, which overlie tilted Santa Fe Group units, and high (²³⁴U/²³⁸U)_iwe infer that the Santa Fe fault has not produced a ground‐rupturing earthquake within the last 490 ± 52 to 592 ± 110 ka (2σ). Our study suggest that travertine formation is driven by fluid flow facilitated by tectonic and mantle structures.