A novel artificial loop scaffold for the noncovalent constraint of peptides.
BACKGROUND
Few examples exist of peptides of < 35 residues that 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 minidomains.


RESULTS
We have examined a novel method for the noncovalent constraint of peptides by attaching the peptide EFLIVKS (single-letter amino acid code), which forms dimers, to the amino and carboxyl termini of different peptide inserts. An 18 residue random coil taken from the inhibitor loop of barley chymotrypsin inhibitor 2 was inserted between the peptides to produce a 32-mer minidomain that is attacked only slowly by elastase, has numerous slowly exchanging protons, contains a high beta-structure content and has a T(m) above 37 degrees C. A point mutation disrupting the hydrophobic interior in both dimerizing peptides causes a loss of all slowly exchanging protons and of secondary structure. Adding specific charged residues to each terminus substantially increased the T(m), as did point mutants designed to add interdimerizer ion pairs. Three flexible epitope tag inserts and a nonamer insert do not appear to be folded in a stable structure by EFLIVKS. The properties of two peptides selected for expression in HeLa cells suggest they do form a stable tertiary structure.


CONCLUSIONS
Attaching short dimerizing peptides to both the amino and carboxyl termini of several 18-mer peptides appears to create stable monomeric tertiary structures. Mutations in the dimerizers can either destabilize or significantly stabilize a standard 18-mer insert. Dimerizing peptides flanking random insert sequences could be used as a strategy to generate heterogeneous peptide libraries with both extended and folded members.
Gururaja
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TL
Rigel, Inc., South San Francisco, CA 94080, USA.
Narasimhamurthy
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S
Payan
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DG
Anderson
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DC
D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D000818:Animals; D002850:Chromatography, Gel; D002942:Circular Dichroism; D003903:Deuterium / Q000378:metabolism; D019281:Dimerization; D004791:Enzyme Inhibitors; D008969:Molecular Sequence Data; D010196:Pancreatic Elastase / Q000037:antagonists & inhibitors / Q000378:metabolism; D019151:Peptide Library; D010455:Peptides / Q000138:chemical synthesis / Q000737:chemistry* / Q000302:isolation & purification / Q000378:metabolism*; D010940:Plant Proteins / Q000737:chemistry* / Q000378:metabolism; D015202:Protein Engineering; D017433:Protein Structure, Secondary; D017434:Protein Structure, Tertiary; D013329:Structure-Activity Relationship
D004791:Enzyme Inhibitors; D019151:Peptide Library; D010455:Peptides; D010940:Plant Proteins; C093336:chymotrypsin inhibitor 2; D003903:Deuterium; D010196:Pancreatic Elastase