ISSN:
0006-3525
Keywords:
protein folding
;
de novo designed protein
;
protein stability
;
structural cassette mutagenesis
;
coiled coil
;
disulfide bridge
;
Chemistry
;
Polymer and Materials Science
Source:
Wiley InterScience Backfile Collection 1832-2000
Topics:
Chemistry and Pharmacology
Notes:
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 α-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 α-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 β-strand cassette from an immunoglobulin fold was inserted. The cassette was fully induced into the α-helical conformation with a [GdnHCl]1/2 value of 3.2M. To demonstrate the importance of short-range interactions (β-sheet/α-helical propensities of amino acid side chains) in modulating structure and stability, a series of 1-5 threonine residues (highest β-sheet propensity) were substituted into the solvent-exposed portions of the cassette in the α-helical conformation. Each successive substitution systematically decreased the stability of the coiled coil with peptide T4b (4 Thr residues) having a [GdnHCl]1/2 value of 2.2M. The single substitution of Ile in the hydrophobic core of the cassette with Ala or Thr had the most dramatic effect on protein stability (peptide I20T, [GdnHCl]1/2 value of 1.4M). Though these substitutions were able to modulate stability, they were not able to disrupt the α-helical conformation of the cassette, showing the importance of the nucleating α-helices on either side of the cassette in controlling conformation of the cassette. We have demonstrated the feasibility of our model protein to accept a β-strand cassette. The effect of cassettes containing other β-strands, β-turns, loops, regions of undefined structure, and helical segments on conformation and stability of our model protein will also be determined. © 1998 John Wiley & Sons, Inc. Biopoly 47: 101-123, 1998
Additional Material:
11 Ill.
Type of Medium:
Electronic Resource
Permalink