A long range goal of my laboratory is to understand the specificity of DNA- protein interaction at a molecular level and to use this knowledge to design peptides and proteins that will recognize predetermined DNA sequences.
Our first aim i s to investigate if a two-helix peptide, similar to the well characterized `helix-turn-helix' motif found in several DNA-binding proteins, is capable of sequence-specific DNA recognition and if such specificities could be altered in a predictable manner. Using the structural information available from biochemical and X-ray crystallographic studies on several DNA-binding proteins, model building and computer graphics, we have designed a two-helix peptide as our standing model. The recognition helix of our model peptide, on its DNA-facing side, contains residues that are known to make sequence-specific contacts with bases in the major groove. In designing the peptide, we have chosen amino acids with a high tendency to form alpha-helices to constitute the helical segments of the peptide and introduced one disulfide linkage and two hydrophobic contacts between the two helices. The peptide also contains basic amino acid residues designed to form nonspecific ionic contacts with the DNA backbone phosphates. Considering possible hydrogen bond donor/acceptor and van der Waals interactions between amino acid side chains and bases in the major groove, we can predict the change in DNA sequence specificities resulting from amino acid substitutions in the peptide. We will make such amino acid substitutions to test our predictions. The model peptide and its variants will be prepared by solid phase peptide synthesis. The binding of these synthetic peptides to DNA's containing the original and predicted sequences will be determined by a variety of methods including a quantitative footprinting technique, providing a direct measure of the binding constant to the specific DNA sequence over and above any nonspecific DNA interaction. Thus we hope to probe DNA-peptide interaction at a chemical level approaching a single base pair change.
The first aim will establish our ability to A priori design peptides which recognize short (6-8 bp) DNA sequences.
Our second aim i s to design, synthesize and test a small protein which recognizes a longer DNA sequence (16bp) with high specificity. Two of the recognition sequences designed in aim 1 will be joined together by a structural helix, the length of which will be adjusted to allow the two recognition helices to interact with two adjacent major grooves on DNA. The protein molecule will be prepared by synthesizing, cloning and expressing a designed gene coding for the protein. The gene will be designed to incorporate several unique restriction sites, making it easy to alter its sequence by cassette mutagenesis. The cloned synthetic gene will be used to make amino acid substitutions in the protein molecule. The knowledge gained will permit the design of new reagents which could recognize specific DNA sequences in vitro, provide a better understanding of protein-DNA interactions as for example in gene regulation and ultimately would permit the in vivo expression of predesigned DNA-binding proteins for both biological studies and medical purposes.
| Aberle, A M; Reddy, H K; Heeb, N V et al. (1994) Stabilization of beta-ribbon structures in peptides using disulfide bonds. Biochem Biophys Res Commun 200:102-7 |
| Forood, B; Feliciano, E J; Nambiar, K P (1993) Stabilization of alpha-helical structures in short peptides via end capping. Proc Natl Acad Sci U S A 90:838-42 |
