Switching DNA-binding specificity by unnatural amino acid substitution.
The specificity of protein-nucleic acid recognition is believed to originate largely from hydrogen bonding between protein polar atoms, primarily side-chain and polar atoms of nucleic acid bases. One way to design new nucleic acid binding proteins of novel specificity is by structure-guided alterations of the hydrogen bonding patterns of a nucleic acid-protein complex. We have used cI repressor of bacteriophage lambda as a model system. In the lambda-repressor-DNA complex, the epsilon-NH(2) group (hydrogen bond donor) of lysine-4 of lambda-repressor forms hydrogen bonds with the amide carbonyl atom of asparagine-55 (acceptor) and the O6 (acceptor) of CG6 of operator site O(L)1. Substitution of lysine-4 (two donors) by iso-steric S-(2-hydroxyethyl)-cysteine (one donor and one acceptor), by site-directed mutagenesis and chemical modification, leads to switch of binding specificity of lambda-repressor from C:G to T:A at position 6 of O(L)1. This suggests that unnatural amino acid substitutions could be a simple way of generating nucleic acid binding proteins of altered specificity.
Maiti
Atanu
A
Department of Biophysics, Bose Institute P-1/12, C.I.T. Scheme VII M, Kolkata 700 054, India.
Roy
Siddhartha
S
D019943:Amino Acid Substitution; D002942:Circular Dichroism; D003545:Cysteine / Q000031:analogs & derivatives* / Q000737:chemistry; D004268:DNA-Binding Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D006860:Hydrogen Bonding; D008239:Lysine / Q000737:chemistry / Q000235:genetics; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed; D009875:Operator Regions, Genetic; D012097:Repressor Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D013050:Spectrometry, Fluorescence; D014764:Viral Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D054334:Viral Regulatory and Accessory Proteins
D004268:DNA-Binding Proteins; D012097:Repressor Proteins; D014764:Viral Proteins; D054334:Viral Regulatory and Accessory Proteins; C016649:phage repressor proteins; C046572:S-(2-hydroxyethyl)cysteine; D008239:Lysine; D003545:Cysteine