Structural cassette mutagenesis in a de novo designed protein: Proof of a novel concept for examining protein folding and stability
✍ Scribed by Stanley C. Kwok; Brian Tripet; Jeff H. Man; Mundeep S. Chana; Pierre Lavigne; Colin T. Mant; Robert S. Hodges
- Publisher
- Wiley (John Wiley & Sons)
- Year
- 1998
- Tongue
- English
- Weight
- 586 KB
- Volume
- 47
- Category
- Article
- ISSN
- 0006-3525
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✦ Synopsis
The solution to the protein folding problem lies in defining the relative energetic contributions of short-range and long-range interactions. In other words, the tendency of a stretch of amino acids to adopt a final secondary structural fold is context dependent. Our approach to this problem is to address whether an amino acid sequence, a ''cassette,'' with a defined secondary structure in the three-dimensional structure of a native protein, can adopt a different conformation when placed into a different protein environment. Thus, we designed de novo a disulfide-bridged two-stranded a-helical parallel coiled coil, where each polypeptide chain consisted of 39 residues, as a ''cassette holder.'' The 11-residue cassette would be inserted into the center of each polypeptide chain between the two nucleating a-helices to replace the control sequence. This Structural Cassette Mutagenesis model permits the analysis of short-range interactions within the inserted cassette as well as long-range interactions between the nucleating helices and the cassette region. The cassette holder, with a control sequence as the cassette, had a GdnHCl transition midpoint during denaturation of 5.6M. To demonstrate the feasibility of our model, an 11-residue b-strand cassette from an immunoglobulin fold was inserted. The cassette was fully induced into the a-helical conformation with a [GdnHCl] 1/2 value of 3.2M. To demonstrate the importance of short-range interactions (bsheet/a-helical propensities of amino acid side chains) in modulating structure and stability, a series of 1-5 threonine residues (highest b-sheet propensity) were substituted into the solvent-exposed portions of the cassette in the a-helical conformation.