Derivatives of CAP Having No Solvent-Accessible Cysteine Residues, or Having a Unique Solvent-Accessible Cysteine Residue at Amino Acid 2 of the Helix-Turn-Helix Motif

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Abstract
The Escherichia coli catabolite gene activator protein (CAP) is a helix-turn-helix motif sequence-specific DNA binding protein. CAP contains a unique solvent-accessible cysteine residue at amino acid 10 of the helix-turn-helix motif. In published work, we have constructed a prototype semi-synthetic site-specific DNA cleavage agent from CAP by use of cysteine-specific chemical modification to incorporate a nucleolytic chelator-metal complex at amino acid 10 of the helix-turn-helix motif [Ebright R., Ebright Y., Pendergrast P.S. and Gunasekera, A., Proc. Natl. Acad. Sci. USA 87, 2882–2886 (1990)]. Construction of second-generation semi-synthetic site-specific DNA cleavage agents from CAP requires the construction of derivatives of CAP having unique solvent-accessible cysteine residues at sites within CAP other than amino acid 10 of the helix-turn-helix motif. In the present work, we have constructed and characterized two derivatives of CAP having no solvent-accessible cysteine residues: [Serl78]CAP and [Leul78]CAP. In addition, in the present work we have constructed and characterized one derivative of CAP having a unique solvent- accessible cysteine residue at amino acid 2 of the helix-turn-helix motif: [Cys170;Ser178]CAP.