Cu-NirK from Haloferax mediterranei as an example of metalloprotein maturation and exportation via Tat system

The green Cu-NirK from Haloferax mediterranei (Cu-NirK) has been expressed, refolded and retrieved as a trimeric enzyme using an expression method developed for halophilic Archaea. This method utilizes Haloferax volcanii as a halophilic host and an expression vector with a constitutive and strong promoter. The enzymatic activity of recombinant Cu-NirK was detected in both cellular fractions (cytoplasmic fraction and membranes) and in the culture media. The characterization of the enzyme isolated from the cytoplasmic fraction as well as the culture media revealed important differences in the primary structure of both forms indicating that Hfx. mediterranei could carry out a maturation and exportation process within the cell before the protein is exported to the S-layer. Several conserved signals found in Cu-NirK from Hfx. mediterranei sequence indicate that these processes are closely related to the Tat system. Furthermore, the N-terminal sequence of the two Cu-NirK subunits constituting different isoforms revealed that translation of this protein could begin at two different points, identifying two possible start codons. The hypothesis proposed in this work for halophilic Cu-NirK processing and exportation via the Tat system represents the first approximation of this mechanism in the Halobacteriaceae family and in Prokarya in general.

Regulation of ammonium assimilation in Haloferax mediterranei: Interaction between glutamine synthetase and two GlnK proteins

GlnK proteins belong to the PII superfamily of signal transduction proteins and are involved in the regulation of nitrogen metabolism. These proteins are normally encoded in an operon together with the structural gene for the ammonium transporter AmtB. Haloferax mediterranei possesses two genes encoding for GlnK, specifically, glnK1 and glnK2. The present study marks the first investigation of PII proteins in haloarchaea, and provides evidence for the direct interaction between glutamine synthetase and both GlnK1 and GlnK2. Complex formation between glutamine synthetase and the two GlnK proteins is demonstrated with pure recombinant protein samples using in vitro activity assays, gel filtration chromatography and western blotting. This protein–protein interaction increases glutamine synthetase activity in the presence of 2-oxoglutarate. Separate experiments that were carried out with GlnK1 and GlnK2 produced equivalent results.

Algorithm for Correcting the Keratometric Error in the Estimation of the Corneal Power in Eyes With Previous Myopic Laser Refractive Surgery

Purpose: To calculate theoretically the errors in the estimation of corneal power when using the keratometric index (nk) in eyes that underwent laser refractive surgery for the correction of myopia and to define and validate clinically an algorithm for minimizing such errors. Methods: Differences between corneal power estimation by using the classical nk and by using the Gaussian equation in eyes that underwent laser myopic refractive surgery were simulated and evaluated theoretically. Additionally, an adjusted keratometric index (nkadj) model dependent on r1c was developed for minimizing these differences. The model was validated clinically by retrospectively using the data from 32 myopic eyes [range, −1.00 to −6.00 diopters (D)] that had undergone laser in situ keratomileusis using a solid-state laser platform. The agreement between Gaussian (PGaussc) and adjusted keratometric (Pkadj) corneal powers in such eyes was evaluated. Results: It was found that overestimations of corneal power up to 3.5 D were possible for nk = 1.3375 according to our simulations. The nk value to avoid the keratometric error ranged between 1.2984 and 1.3297. The following nkadj models were obtained: nkadj= −0.0064286r1c + 1.37688 (Gullstrand eye model) and nkadj = −0.0063804r1c + 1.37806 (Le Grand). The mean difference between Pkadj and PGaussc was 0.00 D, with limits of agreement of −0.45 and +0.46 D. This difference correlated significantly with the posterior corneal radius (r = −0.94, P < 0.01). Conclusions: The use of a single nk for estimating the corneal power in eyes that underwent a laser myopic refractive surgery can lead to significant errors. These errors can be minimized by using a variable nk dependent on r1c.