The OCR protein of bacteriophage T7 is a small, dimeric protein which inhibits the cleavage of the phage DNA by type I restriction enzymes present in the infected host cell. I have studied the structure and the stability of OCR and analysed its binding to the <i>Eco</i>KI type I restriction-modification enzyme. OCR has an unique Tryptophan residue in position 94 which is solvent exposed and important for protein stability but not activity. The protein has a melting temperature of 72.19 °C and a molar extinction coefficient of 32095 M<sup>-1</sup> cm<sup>-1</sup>. Asparagine 4 residue from one monomer is in close proximity to Asparagine 4 in the other monomer. Serine 68 residues are also at the monomer-monomer interface. Six surface exposed amino acids were substituted with cysteine and labelled with cysteine-specific fluorophores. The interaction between labelled OCR(Cys) proteins and <i>Eco</i>KI methylase revealed a huge surface area buried at the interface of the two proteins. OCR binds tightly to the R and S subunits of <i>Eco</i>KI and weakly to the M subunit of <i>Eco</i>KI. One OCR dimer binds to <i>Eco</i>KI methylase and two dimers to <i>Eco</i>KI nuclease. Both OCR-<i>Eco</i>KI methylase and OCR-<i>Eco</i>KI nuclease complexes have a K<sub>d</sub> of about 10<sup>-11<i> </i></sup>M.
| Identifer | oai:union.ndltd.org:bl.uk/oai:ethos.bl.uk:641120 |
| Date | January 2001 |
| Creators | Atanasici, C. |
| Publisher | University of Edinburgh |
| Source Sets | Ethos UK |
| Detected Language | English |
| Type | Electronic Thesis or Dissertation |
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