Figure 1.
Three structural model of the PhaF phasin.
(A) Secondary structure prediction of the N-terminal domain carried out by the PHD (yellow), Jpred (green) and MLRC (cyan) methods. H, alpha-helix; E, extended (beta); c, random-coil. Consensus helices are represented by cylinders. Boldface residues constitute the predicted coiled-coil stretch by the Lupas (red) and MARCOIL (blue) methods, indicating the scheme of the heptad repeats above the sequence. Leucine residues within the coiled-coil are shown underlined and italicized. (B) Helical-wheel representation of the predicted leucine zippers interacting as a parallel tetramer, using DRAWCOIL. (C) Sequence comparison between PhaF (black) and AlgP (blue). Colons represent identical amino acids, and dots indicate conservative changes. (D) Secondary structure prediction of the C-terminal tail. Color scheme as in (A). (E) Joint model structure of monomeric PhaF complexed with DNA. The leucine zipper is shown in blue.
Figure 2.
Prediction of PhaF disordered regions.
Programs used were PONDR-FIT, DISOPRED2, DisEMBL AND DISpro (see Materials and Methods).
Figure 3.
Analytical centrifugation experiments.
(A) Sedimentation coefficient distributions c(s) corresponding to the sedimentation velocity of PhaF. (B) Sedimentation equilibrium analysis of the association state of PhaF. Sedimentation equilibrium absorbance gradients were carried out at 10,000 r.p.m. (open circles) and 16,000 r.p.m. (closed circles). The solid lines show the corresponding best-fit gradients for a single sedimenting species at equilibrium with a solution average molar mass of 85000 Da. The residuals (difference between the experimental data and the fitted data for each point) are shown at the bottom of this panel.
Figure 4.
TFE titrations of PhaF and C-PhaF.
(A) Far-UV CD spectra of PhaF in the absence (solid line) and presence (dashed line) of 40% TFE. (B) Far-UV CD spectra of C-PhaF in the absence (solid line) and presence (dashed line) of 40% TFE. (C) CD-monitored TFE titration of PhaF (closed circles) and C-PhaF (open circles) following the ellipticity signal at 222 nm. (D), same as in (C) but following the signal at 208 nm.
Table 1.
Secondary structure quantitation of PhaF based in CD data.
Figure 5.
(A) Far-UV CD spectra of PhaF at different temperatures. Recovered spectra were registered upon immediate cooling down the scanned sample and after a 30 min waiting period at 20 °C. (B) Near-UV CD spectra of PhaF. (C) Far-UV CD spectra of the isolated C-terminal domain of PhaF (C-PhaF protein). Inset, difference CD spectrum of C-PhaF (20°C–90 °C). (D) CD-monitored temperature scans of PhaF. Solid line indicates fitting of the far-UV CD-monitored transition (protein concentration: 3.8 µM) to the Gibbs-Helmholtz equation (see Materials and Methods). Inset, temperature scan of C-PhaF monitored at 205 nm.
Table 2.
Thermodynamic quantities of the thermal denaturation of PhaFa.
Figure 6.
(A) Far-UV CD spectra of PhaF in the absence (solid line) and the presence (dashed line) of 4.1 M urea. (B). CD-monitored urea titration of 3.8 µM PhaF (closed circles), 11.4 µM PhaF (open triangles) and C-PhaF (open circles). The solid lines represents the fitting to a two-state equilibrium denaturation transition (see text).
Figure 7.
(A) Far-UV CD spectra of PhaF registered at different pH's. (B) pH titration of PhaF monitored by far-UV CD (open circles), average tryptophan fluorescence emission intensity (closed circles) and fluorescence intensity at 320 nm (triangles). (C) Intrinsic tryptophan fluorescence spectra of PhaF at different pH's. (D) Far-UV CD spectra of C-PhaF.
Figure 8.
A model of the interaction between PHA granules, phasins and chromosomal DNA in Pseudomonas putida KT2440.
A PhaF tetramer is depicted interacting with the PHA granule while also attached to a fragment of nucleoid DNA. The hydrophobic residues in the N-terminal domain are shown in orange, while the polar ones are colored blue. The leucine zipper is colored green, and the DNA is depicted in cyan. Figure was rendered with RASTOP 2.2 (http://www.geneinfinity.org/rastop/) only for visualization purposes, so that no atomic detail is intended.