Toward the design of singlechain proteins

To facilitate the formation of stable native-like de novo proteins, the lessons learned from the modular approach were applied to the design of single-chain proteins. This was accomplished by covalently linking together the individual elements of secondary structure (Figure 11.1B). In pioneering work, DeGrado and coworkers linked two aj peptides with a short turn sequence, forming a helix-turn-helix (denoted a2), and showed that two a2 sequences could dimerize to form a four-helix bundle (Ho and DeGrado 1987). The DeGrado group proceeded to the final step of producing a single chain four-helix bundle by linking the two a2 peptides to form a single a4 chain. The resulting 74-amino-acid sequence folded into a stable a-helical bundle (Regan and DeGrado 1988).

The four helices in a4 were identical to one another and composed of only three amino acids—Glu, Lys, and Leu. The next step in the bottom-up approach required the design of a single-chain protein with a nonrepeating sequence. Hecht and coworkers (1990) achieved this goal by designing Felix, a novel four-helix bundle protein containing 19 of the 20 natural amino acids arranged in four different a-helices. As was done for the preceding designed helical bundles, the a-helices of Felix were designed to be amphiphilic. Special attention was given to the role of position-dependent a-helical propensities (Richardson and Richardson 1988) and further stabilization of the structure was achieved by designing a single intramolecular disulfide bond between the first and fourth helices. Moreover, to favor the desired structure, "negative design" was used to destabilize competing structures: Thus, alternating polar and nonpolar residues, which would favor p-strands (West and Hecht 1995), were avoided, turns were designed to disfavor the continuation of one long helix, and the hydrophobic faces of the helices were designed to favor a "left-turning" bundle topology (Hecht et al. 1990). The resulting protein was shown to fold into a globular helical structure. However, it was not very stable, and like a4, it displayed molten globule-like characteristics. For both Felix and a4, the dynamic nature of the structures presumably resulted from nonunique, poorly packed hydrophobic cores.

0 0

Post a comment