Question

1. (20 pts) Reduction of the disulfide bond in human PrP91-231 (prion protein) and lowering the pH to 4.0, in a dilute acetate buffer generated a highly soluble monomeric protein. Figure 1. Determination of the apparent molecular weight of PrP by size-exclusion chromatography. (A) Elution profile of standards used to construct a calibration curve of molecular weight (MW) versus elution time. (B) Oxidized human PrP (native form) (pH 8.0) elutes with an apparent MW of 18 kD. (C) Reduced human PrP (pH 4.0). (D) Oxidized human PrP (pH 4.0) treated with 1 M GuHCi 158 kD 1.4 kD 670 kD 44 kD 17 kD A 18 kD C 1.0 Ab Units 200 1000 400 600 800 Retention time (s) Figure 2. Structure of the PrP isoforms. (A) Far-UV CD spectra of Oxidized human PrP (pH 8.0) (0) and reduced human PrP (pH 4.0) (). (B) Near-UV CD spectra for oxidized human PrP (pH 8.0) (o), reduced human PrP (pH 4.0) (), and GuHCl-treated human PrP () A 20 B 20 15 10 -20 -40 -5 10 -60 250 260 270 280 290 300 310 15 230 240 200 210 220 250 Wavelength (nm) understanding of the above experiments Wavelength (nm) Answer the following questioi (a) Determine the size of both reduced human PrP and GuHCl-treated human PrP. (Hint: The accuracy of the size of the proteins depends on the accuracy of your measurement of the protein mobility) (b) Comment on the secondary structures of the oxidized human PrP and reduced human PrP (c) Comment on the tertiary structures of the oxidized human PrP, reduced human PrP and the GuHCl treated human PrP (d) Based on your answers in (a) to (c), suggest what might have happened to the structure of oxidized human PrP (pH 4.0) after being treated with 1 M GuHCI Absorption at 280 nm Molecular ellipticity Molecular ellipticity m

Answer 1

Ans b. Analyse the figure 2A carefully. Oxidised form of prion protein has 2 dips at 210 nm and 220 nm. This peak is the characteristic peak for alpha helical structure of a protein. This means oxidised form has Alpha helix as the secondary structure due to lack of disulfide linkages.

On the other hand, reduced form of prion protein has only single dip around 215 nm which is the characteristic of ß sheet structure. This suggests that with intact  disulfide bond and loss of some hydrophobic interactions at low pH results in different arrangement of amino acid residues resulting in ß Sheet structure.

Ans c. Tertiary structure of the protein is the 3-D structure which occurs due to various interactions among the amino acid residues like ionic interactions, hydrophobic Bonds, Hydrogen bonding and disulfide linkages. Oxidised form is more compactly folded than reduced form. It's aromatic amino acid residues are buried in the domains. While GuHCl treated prion protein will lose its tertiary structure by breaking all hydrophobic interactions and make the protein denature.