Helix propagation of the S-peptide sequence (residues 1-19 of ribonuclease A) in 2,2,2-trifluoroethanol (TFE) solutions has been investigated with CD and nmr Overhauser effect spectroscopies. In this study, the S-peptide helix is covalently initiated at the N-terminus through disulfide bonds to a helix scaffold derived from the N-terminal sequence of the bee venom peptide apamin. The entire S-peptide sequence of this hybrid sequence peptide becomes helical at high proportions of TFE. Residues 14-19 of the S-peptide are not helical in the free peptide in TFE, nor are they helical in ribonuclease A. The "helix stop" signal encoded by the S-peptide sequence near residue 13 does not persist at high TFE with this hybrid sequence peptide. The helix-stabilizing effects of TFE are due at least in part to facilitated propagation of an extant helix. This stabilizing effect appears to be a general solvation effect and not due to specific interaction of the helical peptide with TFE. Specifically these data support the idea that TFE destabilizes the coil state by less effective hydrogen bonding of the peptide amide to the solvent.

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Quantification of apoptotic cell death in vivo has become treatment (r 2 ! .947, N ! 27 samples). There was also a direct temporal relationship between an increase in the CH 2 / an important area of investigation in patients with acute lymphoblastic leukemia (ALL). We have devised a noninva-CH 3 signal intensity ratio and the onset of apoptosis as detected by nuclear morphologic analysis, fluorescein-annexin sive analytical method to estimate the percentage of apoptotic lymphoblasts in doxorubicin-treated Jurkat T-cell ALL V flow cytometry, and DNA gel electrophoresis. Thin-layer chromatography confirmed that a dynamic and/or composi-cultures, using proton nuclear magnetic resonance spectroscopy ( 1 H NMR). We have found that the ratio of the methyl-tional change of the plasma membrane, rather than increases in lipase activity or fatty acid production, appears ene (CH 2 ) resonance (at 1.3 ppm) to the methyl (CH 3 ) resonance (at 0.9 ppm) signal intensity, as observed by 1 H NMR, to account for the increase in the CH 2 /CH 3 signal intensity ratio during apoptosis. 1 H NMR may have clinical utility for is directly proportional to the percentage of apoptotic lymphoblasts in vitro. The correlation between the CH 2 /CH 3 the early noninvasive assessment of chemotherapeutic efficacy in patients with ALL. signal intensity ratio and the percentage of apoptotic lymphoblasts was optimal 24 to 28 hours after doxorubicin ᭧

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...