Including solvation effects (in the Poisson-Boltzmann continuum solvent approximation) we report ab initio quantum mechanical calculations (HF/6-31G**) on the conformational energies for adding alanine to the amino or carboxyl terminus of a polyalanine R-helix as a function of helix length N. We find that extending the length of an R-helix increasingly favors the R-helix conformation for adding an additional residue, even in hydrophobic environment. Thus, R-helix formation is a cooperative process. Using charges from the QM calculations, we find that the electrostatic energy dominates the QM results, showing that this increasing preference for R-helix formation results from dipole-dipole interaction within the R-helix. These results provide quantitative preferences and insight into the conformational preferences and kinetics of protein folding.

It is generally believed that leucine zipper regulatory proteins for DNA transcription recognize their DNA binding sites as dimers preformed in solution (and that the monomers do not bind specifically to these sites). To test this idea, we synthesized the 31-residue peptide v-Jun-br, which contains only the DNA binding region of the v-Jun monomer. Footprinting assays show that v-Jun-br monomers specifically protect the DNA binding site of v-Jun in almost identically the same way as dimers. Thus, (i) the monomer recognizes the half-site of the dimer binding site and (ii) dimerization does not appreciably affect the bound conformation of each monomer. These results may have implications in the regulation of transcription by such proteins. Thus, two monomers of v-Jun might bind sequentially to the dimer binding site followed by dimerization of v-Jun while bound. This may allow binding at concentrations too low for dimerization in solution.

We present a general strategy for dsgning proteins to recognize DNA sequences and illustrate this with an example based on the "Y-shaped scissors grip" model for leucine-zipper gene-regulatory proteins. The designed protein is formed from two copies, in tandem, of the basic (DNA binding) region of v-Jun. These copies are coupled through a tripeptide to yield a "dimer" expected to recognize the sequence TCATCGATGA (the v-Jun-v-Jun homodimer recognizes ATGACTCAT). We synthesized the protein and oligonucleotides containing the proposed binding sites and used gel-retardation assays and DNase I footprinting to esablh that the dimer binds specifically to the DNA sequence TCATC-GATGA but does not bind to the wild-type DNA-sequences, nor to oligonucleotides in which the recognition half-site is modifled by single-base changes. These results also provide strong support for the Y-shaped scissors grip model for binding of leucine-zipper proteins.We propose a general strategy for designing proteins to recognize specific DNA-binding sites: this strategy is to select segments of proteins, each of which recognizes particular DNA segments and to stitch these segments together via a short peptide with a cystine crosslink in a way compatible with each peptide being able to bind to its own DNA segment. This technique creates a protein that recognizes the composite site.As a starting point we consider the gene-regulatory leucinezipper proteins. They are characterized by two structural motifs (1-3): (i) the leucine zipper, which is responsible for dimerization, and (ii) the basic region for DNA binding (4-7). The basic regions of unbound leucine-zipper dimers are unfolded but fold into the a-helix conformation upon binding to the specific site (8-10). The most plausible model for the conformation of leucine-zipper protein is the "Y-shaped scissors grip" model (1, 2), in which the basic region of each monomer interacts with DNA on either side of the dyad axis of the binding site. Thus, for yeast transcriptional activator GCN4 each arm recognizes the half-site AGTA (11,12). DESIGN Our design strategy assumes this Y-shaped scissors grip model (Fig. la). We design proteins by crosslinking (stitching together) various binding arms so as to be consistent with the orientation of the recognition helix in each half-site. Here we build upon the results of Kim and coworkers (5, 6), who showed that the leucine zipper of GCN4 can be replaced with linkers (Gly-Gly-Cys) at the C terminus of the DNA binding segment, which upon oxidation dimerize and bind to the same site (ATGACTCAT) as GCN4. As a model system to explore the design of additional DNA-binding proteins, we have chosen the v-Jun leucine-zipper dimer (Fig. la), which also binds to the site ATGACTCAT as a homodimer with itself or as a heterodimer with Fos (4, 13-16), another member of this DNA-binding protein family. We will reverse the sequence relationship of the a-helix to the target nucleotide of the binding arms by adding the Gly-Gly-Cys linker to the N terminus (rather than ...

Using protein stitchery with appropriate

No abstract