RNA-Activated Protein Kinase, PKR

The double-stranded RNA (dsRNA) activated protein kinase, PKR, is one of the several enzymes induced by interferons and a key molecule mediating the antiviral effects of interferons. PKR contain an N-terminal, double-stranded RNA binding domain (dsRBD), which has two tandem copies of the motifs (dsRBM I and dsRBM II). Upon binding to viral dsRNA, PKR is activated via autophosphorylation. Activated PKR has several substrates; one of the examples is eukaryotic translation initiation factor 2 (eIF2a). The phosphorylation of eIF2a leads to the termination of cell growth by inhibiting protein synthesis in response to viral infection. The objective of this project was to characterize the dsRBM I and define the dsRNA binding using biophysical methods. First, the dsRBM I gene was cloned from a pET-28b to a pET-11a expression plasmid. N-terminal poly-histidine tags on pET-28b are for affinity purification; however, these tags can alter the structure and function of proteins, thus the gene of dsRBM I was transferred into the plasmid without tags (pET-11a) and expressed as a native protein. The dsRBM I was transformed into and expressed by Rosetta DE3plyS expression cells. Purification was done by FPLC using a Sepharose IEX ion exchange followed by Heparin affinity column; yielding pure protein was assayed by PAGE. Analytical Ultracentrifugation, Sedimentation Velocity, was used to characterize free solution association state and hydrodynamic properties of the protein. The slight decrease in S-value with concentration is due to the hydrodynamic non-ideality. No self association was observed. The obtained molecule weight was 10,079 Da. The calculated sedimentation constant at zero concentration at 20°C in water was 1.23 and its friction coefficient was 3.575 ´ 10-8. The frictional ratio of sphere and dsRBM I became 1.30. Therefore, dsRBM I must be non-globular and more asymmetric shape. Isolated dsRBM I exhibits the same tertiary fold as compared to context in the full domain but it exhibited weaker binding affinity than full domain to a 20 bp dsRNA. However, when the conditions allowed for its saturation, dsRBM I to 20 bp dsRNA has similar stoichiometry as full dsRBD