A Ratiometric Fluorescent Probe for Imaging of the Activity of Methionine Sulfoxide Reductase†A in Cells.

Methionine sulfoxide reductase A (MsrA) is an enzyme involved in redox balance and signaling, and its aberrant activity is implicated in a number of diseases (for example, Alzheimer's disease and cancer). Since there is no simple small molecule tool to monitor MsrA activity in real time in vivo, we aimed at developing one. We have designed a BODIPY-based probe called (S)-Sulfox-1, which is equipped with a reactive sulfoxide moiety. Upon reduction with a model MsrA (E. coli), it exhibits a bathochromic shift in the fluorescence maximum. This feature was utilized for the real-time ratiometric fluorescent imaging of MsrA activity in E. coli cells. Significantly, our probe is capable of capturing natural variations of the enzyme activity in vivo.

Crystallization of nepenthesin I using a low-pH crystallization screen.

Nepenthesins are aspartic proteases secreted by carnivorous pitcher plants of the genus Nepenthes. They significantly differ in sequence from other plant aspartic proteases. This difference, which provides more cysteine residues in the structure of nepenthesins, may contribute to their unique stability profile. Recombinantly produced nepenthesin 1 (rNep1) from N. gracilis in complex with pepstatin A was crystallized under two different crystallization conditions using a newly formulated low-pH crystallization screen. The diffraction data were processed to 2.9 and 2.8 Šresolution, respectively. The crystals belonged to space group P212121, with unit-cell parameters a = 86.63, b = 95.90, c = 105.40 Å, α = ß = γ = 90° and a = 86.28, b = 97.22, c = 103.78 Å, α = ß = γ = 90°, respectively. Matthews coefficient and solvent-content calculations suggest the presence of two molecules of rNep1 in the asymmetric unit. Here, the details of the crystallization experiment and analysis of the X-ray data are reported.

Expression and characterization of plant aspartic protease nepenthesin-1 from Nepenthes gracilis.

Carnivorous plants of the genus Nepenthes produce their own aspartic proteases, nepenthesins, to digest prey trapped in their pitchers. Nepenthesins differ significantly in sequence from other aspartic proteases in the animal or even plant kingdoms. This difference, which also brings more cysteine residues into the structure of these proteases, can be a cause of uniquely high temperature and pH stabilities of nepenthesins. Their detailed structure characterization, however, has not previously been possible due to low amounts of protease present in the pitcher fluid and also due to limited accessibility of Nepenthes plants. In the present study we describe a convenient way for obtaining high amounts of nepenthesin-1 from Nepenthes gracilis using heterologous production in Escherichia coli. The protein can be easily refolded in vitro and its characteristics are very close to those described for a natural enzyme isolated from the pitcher fluid. Similarly to the natural enzyme, recombinant nepenthesin-1 is sensitive to denaturing and reducing agents. It also has maximal activity around pH 2.5, shows unusual stability at high pH and its activity is not irreversibly inhibited even after prolonged incubation in the basic pH range. On the other hand, temperature stability of the recombinant enzyme is lower in comparison with the natural enzyme, which can be attributed to missing N-glycosylation in the recombinant protein.