Interestingly, the chlamydial vacuole of GR10, a cPDF-overexpressing mutant[11], has different morphology, compared to that of the parental strain. However, there is no stringent sequence requirement for this region for cPDF enzyme 2-Hydroxybenzyl alcohol activity. (contamination exhibit only mild or even no symptoms, therefore, they do not 2-Hydroxybenzyl alcohol seek medical treatment. However, a significant proportion of the untreated patients develop severe complications such as pelvic inflammatory disease and infertility[10]. In the absence of an effective vaccine, chemopreventive steps are being sought after. Since re-infection is very common, as a result of the presence of multiple serovars and the inability of the human body to mount lasting protective immunity against the pathogens, chemoprevention needs to be implemented among the sexually active populace as long as they practice unprotected sex. For this reason, only antichlamydials with little or no effects on other microbial species and the human host should be used for the prevention of sexually transmitted chlamydial contamination. PDF is also essential for PDF using the Modeller (9v5) program[14]. The following PDF structures were used as templates: 1RL4A of (to remove bad steric contacts. The model was solvated in a truncated octahedral periodic box of SPC/E water[23], with the overall charge of the system neutralized by insertion of the appropriate number of sodium ions. A 1-nm short-range cutoff was used for all computations. Long-range electrostatics in the model were treated using the particle mesh Ewald method[24,25]. Each system was subjected to energy minimization of the solvent only, followed by minimization of the entire system before dynamics. A 2-fs time step was used for all Rabbit Polyclonal to EPHB1 molecular dynamics work. All bonds to hydrogen were restrained using the shake method[26]. A preliminary dynamics step restraining motion of the protein while gradually increasing heat from 0 K to 300 K was run for 100 ps. This step was followed by a constant heat (300 K) and constant pressure (1 bar) production run for a time period of 15 ns. As shown in Figure ?Determine3,3, at the end of this 15-ns period, the protein backbones of both wild-type cPDF and the F134C/R137S mutant had fully stabilized. Structural averages of the proteins were calculated from the last 3 ns of simulation and were refined using energy minimization. The average per residue heat factors were calculated from atomic root mean square fluctuation data obtained from the last 2 ns of simulation trajectory. Open in a separate window Physique 3 Molecular dynamics protein backbone atom root mean square deviation plot. Construction and production of recombinant cPDF A pET21-based expression vector for wild-type cPDF carrying a carboxyl terminal (His)6-tag has been previously described[11]. This vector was used as the template for the construction of the D68R, E70R, D68R/E70R or D68-V72 cPDF variants using a QuickChange site-directed mutagenesis kit (Stratagene). Sequence authenticity of the cPDF-coding sequence in the vectors was verified by automated DNA sequencing. Production and purification of the recombinant proteins were carried out following published procedures with modifications[11]. Plasmid-transformed ArcticExpress was cultured on a shaker at 30C. When the A600 of the culture reached about 0.8, the culture temperature was lowered to 13C, isopropyl -D-1-thiogalactopyranoside (final concentration: 1 mmol/L) and CoCl2 (final concentration: 100 mol/L) were added to the culture to induce cPDF gene transcription and subsequent synthesis of cobalt-containing cPDF enzyme. After overnight culture at 13C, the bacteria were collected by centrifugation, and lysed by a French Press. Cell debris was removed by centrifugation at 25?000 for 30 min. cPDFs were purified with the Talon affinity metal Agarose (Clontech) and stored at -80C after the addition of glycerol to a final 2-Hydroxybenzyl alcohol concentration of 10%. PDF activity assay An assay previously developed for. The E70R mutant showed only slightly decreased efficiency. loop region is involved in a rate-limiting conformational change of the enzyme during catalysis. However, there is no stringent sequence requirement for this region for cPDF enzyme activity. (infection exhibit only mild or even no symptoms, therefore, they do not seek medical treatment. However, a significant proportion of the untreated patients develop severe complications such as pelvic inflammatory disease and 2-Hydroxybenzyl alcohol infertility[10]. In the absence of an effective vaccine, chemopreventive measures are being sought after. Since re-infection is very common, as a result of the existence of multiple serovars and the inability of the human body to mount lasting protective immunity against the pathogens, chemoprevention needs to be implemented among the sexually active population as long as they practice unprotected sex. For this reason, only antichlamydials with little or no effects on other microbial species and the human host should be used for the prevention of sexually transmitted chlamydial infection. PDF is also essential for PDF using the Modeller (9v5) program[14]. The following PDF structures were used as templates: 1RL4A of (to remove bad steric contacts. The model was solvated in a truncated octahedral periodic box of SPC/E water[23], with the overall charge of the system neutralized by insertion of the appropriate number of sodium ions. A 1-nm short-range cutoff was used for all computations. Long-range electrostatics in the model were treated using the particle mesh Ewald method[24,25]. Each system was subjected to energy minimization of the solvent only, followed by minimization of the entire system before dynamics. A 2-fs time step was used for all molecular dynamics work. All bonds to hydrogen were restrained using the shake method[26]. A preliminary dynamics step restraining motion of the protein while gradually increasing temperature from 0 K to 300 K was run for 100 ps. This step was followed by a constant temperature (300 K) and constant pressure (1 bar) production run for a time period of 15 ns. As shown in Figure ?Figure3,3, at the end of this 15-ns period, the protein backbones of both wild-type cPDF and the F134C/R137S mutant had fully stabilized. Structural averages of the proteins were calculated from the last 3 ns of simulation and were refined using energy minimization. The average per residue temperature factors were calculated from atomic root mean square fluctuation data obtained from the last 2 ns of simulation trajectory. Open in a separate window Figure 3 Molecular dynamics protein backbone atom root mean square deviation plot. Construction and production of recombinant cPDF A pET21-based expression vector for wild-type cPDF carrying a carboxyl terminal (His)6-tag has been previously described[11]. This vector was used as the template for the construction of the D68R, E70R, D68R/E70R or D68-V72 cPDF variants using a QuickChange site-directed mutagenesis kit (Stratagene). Sequence authenticity of the cPDF-coding sequence in the vectors was verified by automated DNA sequencing. Production and purification of the recombinant proteins were carried out following published procedures with modifications[11]. Plasmid-transformed ArcticExpress was cultured on a shaker at 30C. When the A600 of the culture reached about 0.8, 2-Hydroxybenzyl alcohol the culture temperature was lowered to 13C, isopropyl -D-1-thiogalactopyranoside (final concentration: 1 mmol/L) and CoCl2 (final concentration: 100 mol/L) were added to the culture to induce cPDF gene transcription and subsequent synthesis of cobalt-containing cPDF enzyme. After overnight culture at 13C, the bacteria were collected by centrifugation, and lysed by a French Press. Cell debris was removed by centrifugation at 25?000 for 30 min. cPDFs were purified with the Talon affinity metal Agarose (Clontech) and stored at -80C after the addition of glycerol to a final concentration of 10%. PDF activity assay An assay previously developed for the PDF[27] was modified to measure the activity of the chlamydial enzyme. The assay mix, in a total of 50 L reaction volume, contained 50 mmol/L HEPES (pH 7.2), 10 mmol/L NaCl, 125 ng PDF, 0-50 mmol/L formyl-Met-Ala-Ser (fMAS) and 50-500 nmol/L GM6001. The deformylation reaction was allowed to proceed at 37C for 10 min and then terminated by heating at 95C for 2 min. The amount of MAS produced was reported by 2,4,6.
Interestingly, the chlamydial vacuole of GR10, a cPDF-overexpressing mutant[11], has different morphology, compared to that of the parental strain