Folding and activity of hybrid sequence, disulfide-stabilized peptides.

Abstract

Peptides have been synthesized that have hybrid sequences, partially derived from the bee venom peptide apamin and partially from the S peptide of ribonuclease A. The hybrid peptides were demonstrated by NMR spectroscopy to fold, forming the same disulfides and basic three-dimensional structure as native apamin, containing a f-turn and an a-helix.These hybrids were active in complementing S protein, reactivating nuclease activity. In addition, the hybrid peptide was effective in inducing antibodies that cross-react with the RNase, without conjugation to a carrier protein. The stability of the folded structure of this peptide suggests that it should be possible to elicit antibodies that will react not only with a specific sequence, but also with a specific secondary structure. Hybrid sequence peptides also provide opportunities to study separately nucleation and propagation steps in formation of secondary structure. We show that in S peptide the a-helix does not end abruptly but rather terminates gradually over four or five residues. In general, these hybrid sequence peptides, which fold predictably because of disulfide bond formation, can provide opportunities for examining structure-function relationships for many biologically active sequences.It is well known that in disulfide cross-linked proteins the relative positions of cysteines are strongly conserved. Among families of proteins containing similar disulfides, there tends to be a strong conservation of the threedimensional structure even when the overall sequence homology is rather low (1). Thus, it is clear that a wide variety of amino acid sequences can be accommodated within the fold defined by the cystine bridges. We have taken advantage of this adaptability to form structured, hybrid sequence peptides. In these hybrids the folding is largely controlled by formation of disulfides involving cysteines with relative positions taken from a natural peptide. However, many of the noncysteine residues can be replaced with a sequence from another protein. In the cases discussed here, the disulfides were derived from apamin (2-4), and the noncysteine amino acids from the helical region of apamin were replaced with those from the S peptide of RNase A (5). The S peptide, consisting of the first 20 amino acids from RNase A, has been extensively characterized and has been shown to have a helix-forming propensity (6-8) (existing as up to =40wo helix under optimal conditions). In addition, there was shown to be a helix stop signal present (9, 10) near the 13th or 14th residue. This signal persists even in trifluoroethanol, where the overall helical fraction is significantly increased (11, 12). The S peptide binds (with an association constant of 106) to S protein, the large fragment of RNase A from which it was cleaved, reactivating nuclease activity (3). In the apamin-S peptide hybrids, we show that the disulfides stabilize the helical segment at the beginning of the S peptide completely, giving us the opportunity to investigate the propagation ofthe helix af...

Structures and related properties of helical, disulfide-stabilized peptides

“…The linear least squares fit of the data from 2SOC to 7 5°C is also shown. The typeS of connectivities observed are exactly the same as those in both apamin (Pease & Wemmer, 1988) and the Apa-S hybrid (Pease, et al, 1990) for the first 16 residues at 0°C and 25°C. These include the aH-NH-NH-NH connectivity characteristic of the type I 13-turn, and i,i+2 connectivity at the beginning of the helix, and the continued NH-NH connectivities in the a-helix from residue 9 through residue 16.…”

“…It has been shown that a disulfide framework, taken from the bee venom peptide apamin, can be used to make hybrid sequence peptides with defmed conformations and high stability (Pease, et al, 1990). This chapter describes the application of.this approach to make a hybrid sequence peptide.…”

“…·sequence peptides, and seems to be indicative of the folded core structure (Pease, et al, 1990). The e222 observed corresponds to approximately 42% of the peptide being helical at 25°C (Johnson & Tinoco, 1972).…”

Model Peptide Studies Demonstrate That Amphipathic Secondary Structures Can Be Recognized by the Chaperonin GroEL (cpn60)

“…2); the presence of a helical conformation is confirmed by the small values of 3 J HN-H␣ for these residues. The data for residues [17][18][19] are not indicative of a well formed helix, suggesting that they form a frayed terminus to the helix or adopt a turn conformation. These findings are in line with the structure modeled for APA14 and are consistent with the three-dimensional structures determined for other apamin peptides (35).…”

“…These disulfides lock the COOH-terminal half of apamin (residues 9 -16) into a helical conformation (18,35). Various sequences can be substituted into the COOH terminus of apamin and forced into a helical conformation by the presence of these disulfide bridges (19). 1 The apamin framework can be used to present either the hydrophobic or hydrophilic face of an amphipathic helix (17), and thus it serves as a useful tool for examining the structural requirements for binding to cpn60.…”