Figure 3
Molecular architecture and RNA binding activity of PiCypA.
(a) β-barrel like fold containing eight β-strands and two helices forming cap at either ends (b) aromatic (cyan) and aliphatic (magenta) forming hydrophobic core (c) Residues showing unusual chemical shifts (Magenta) in NMR spectra and aromatic residues (cyan) responsible for it. (d) Western blot of PiCypA protein probed with anti-his antibody (upper panel) as loading control. For this 1 μg of BSA and increasing concentration of PiCypA (1, 2 and 4 μg) proteins were dot-blotted on precharged PVDF membrane. An identical blot was probed with a 13 mer radio labeled RNA oligo (5′-AUAGCCUCAACCG-3′) and developed. (e) Isothermal titration calorimetry indicating hyperbolic nature for PiCypA binding to the 13 mer RNA (f) Selected regions from the superimposed 2D [15N, 1H] HSQC spectra of free and complexed PiCypA shown in red and blue, respectively. The resonance assignments of residues with large chemical shift changes upon RNA binding (1:1 molar ratio) are shown with arrows. (g) Combined chemical shift changes of PiCypA upon RNA binding in 1:1 molar ratio. Red line indicates one standard deviation for the mean chemical shift change of the dataset. Combined chemical shift changes were calculated by taking the square root of the sum of the squares of the chemical shift changes for the backbone 1HN and 15N resonances after scaling the 15N shifts by 0.2 (h) Mapped RNA binding region (R1, R2 and R3) on the crystal structure of PiCypA.
