A novel artificial loop scaffold for the noncovalent constraint of peptides.

Gururaja TL, Narasimhamurthy S, Payan DG, Anderson DC.

Chem Biol 2000 Jul;7(7):515-27.

Background: Few examples exist of peptides of less than 35 residues which form a stable tertiary structure without disulfide bonds. A method for stabilization and noncovalent constraint of relatively short peptides may allow the construction and use of intracellular peptide libraries containing protein mini-domains.

Results: We have examined a novel method for the noncovalent constraint of peptides by attaching the peptide EFLIVKS, which forms dimers, to the N- and C-terminus of different peptide inserts. One insert consists of 18 residues taken from the inhibitor loop of barley chymotrypsin inhibitor 2. The resulting 32mer mini-domain is attacked only slowly by elastase, has numerous slowly-exchanging protons, contains a high beta-structure content when examined by circular dichroism, and has a Tm above 37°C. The 18mer insert alone is a random coil. A specific point mutation disrupting the hydrophobic interior in both dimerizing peptides of the 32mer causes a loss of all slowly exchanging protons and of secondary structure. Adding specific charged residues to each terminus of the 32mer construct substantially increases the Tm. Point mutants designed to add inter-dimerizer ion pairs also significantly increased the Tm. Three flexible epitope tag inserts and a 9mer insert do not appear to be folded in a stable structure by EFLIVKS as they do not have slowly-exchanging protons. However two peptides selected for expression in HeLa cells have slowly exchanging protons, a defined secondary structure, and Tm's of 40.6 and 60°C. They thus may form a stable tertiary structure.

Conclusions: Attachment of short dimerizing peptides to both the N- and C-terminus of several different 18mer peptides appears to create stable monomeric tertiary structures. This is not true for all test sequences nor for a shorter insert. Mutations in the dimerizers can either destabilize or significantly stabilize a standard 18mer insert used here. Use of these dimerizing peptides flanking random insert sequences may be a feasible strategy to generate heterogeneous peptide libraries with both extended and folded members.