NMR Studies on Structure and Dynamics of the Monomeric Derivative of BS-RNase: New Insights for 3D Domain Swapping

Three-dimensional domain swapping is a common phenomenon in pancreatic-like ribonucleases. In the aggregated state, these proteins acquire new biological functions, including selective cytotoxicity against tumour cells. RNase A is able to dislocate both N- and C-termini, but usually this process requires denaturing conditions. In contrast, bovine seminal ribonuclease (BS-RNase), which is a homo-dimeric protein sharing 80% of sequence identity with RNase A, occurs natively as a mixture of swapped and unswapped isoforms. The presence of two disulfides bridging the subunits, indeed, ensures a dimeric structure also to the unswapped molecule. In vitro, the two BS-RNase isoforms interconvert under physiological conditions. Since the tendency to swap is often related to the instability of the monomeric proteins, in these paper we have analysed in detail the stability in solution of the monomeric derivative of BS-RNase (mBS) by a combination of NMR studies and Molecular Dynamics Simulations. The refinement of NMR structure and relaxation data indicate a close similarity with RNase A, without any evidence of aggregation or partial opening. The high compactness of mBS structure is confirmed also by H/D exchange, urea denaturation, and TEMPOL mapping of the protein surface. The present extensive structural and dynamic investigation of (monomeric) mBS did not show any experimental evidence that could explain the known differences in swapping between BS-RNase and RNase A. Hence, we conclude that the swapping in BS-RNase must be influenced by the distinct features of the dimers, suggesting a prominent role for the interchain disulfide bridges

Behavior of chlorpyrifos and its major metabolite TCP (3,5,6-trichloro-2-pyridinol) in agricultural soils amended with drinking water treatment residuals

Purpose: Chlorpyrifos can be effectively adsorbed by drinking water treatment residuals (WTR), ubiquitous and non-hazardous by-products of potable water production. The major metabolite 3,5,6-trichloro-2-pyridinol (TCP) was found to be much more mobile and toxic than its parent chlorpyrifos. To assess the feasibility of WTR amendment for attenuation of chlorpyrifos and TCP pollution, the sorption/desorption and degradation behavior of chlorpyrifos and TCP in WTR-amended agricultural soils was examined in the present study. Materials and methods: Two representative agricultural soils were sampled from southern and northern China, respectively. The soils were amended with WTR at the rates of 0, 2, 5, and 10 % (w/w). Batch sorption/desorption test were applied to investigate the sorption/desorption characteristics of chlorpyrifos and TCP in WTR-amended soils. The influence of WTR amendment on chlorpyrifos degradation and TCP formation was evaluated using the incubation test, and its effect on the soil bacterial abundance was further studied through DNA extraction and PCR amplification. Results and discussion: Results showed that WTR amendment (0–10 %, w/w) significantly enhanced the retention capacity of chlorpyrifos and TCP in both soils examined (P &lt; 0.05). Fractionation analyses further demonstrated that the bioavailability of chlorpyrifos was considerably reduced by WTR amendment, resulting in a decreased chlorpyrifos degradation rate. The WTR amendment also significantly reduced the mobility of TCP formed in chlorpyrifos-contaminated soils (P &lt; 0.001). The chlorpyrifos toxicity to soil bacteria community was largely mitigated following WTR amendment, resulting in increased total bacterial abundance. Conclusions: Results obtained in the present study indicate a great deal of potential for the beneficial reuse of WTR as soil amendments for chlorpyrifos and TCP pollution control.</p