Tankyrase, a protein with homology to ankyrins and to the catalytic domain of poly(adenosine diphosphate-ribose) polymerase (PARP), was identified and localized to human telomeres. Tankyrase binds to the telomeric protein TRF1 (telomeric repeat binding factor-1), a negative regulator of telomere length maintenance. Like ankyrins, tankyrase contains 24 ankyrin repeats in a domain responsible for its interaction with TRF1. Recombinant tankyrase was found to have PARP activity in vitro, with both TRF1 and tankyrase functioning as acceptors for adenosine diphosphate (ADP)-ribosylation. ADP-ribosylation of TRF1 diminished its ability to bind to telomeric DNA in vitro, suggesting that telomere function in human cells is regulated by poly(ADP-ribosyl)ation.

Designed peptides that fold autonomously to specific conformations in aqueous solution are useful for elucidating protein secondary structural preferences. For example, autonomously folding model systems have been essential for establishing the relationship between ␣-helix length and ␣-helix stability, which would be impossible to probe with ␣-helices embedded in folded proteins. Here, we use designed peptides to examine the effect of strand length on antiparallel ␤-sheet stability. ␣-Helices become more stable as they grow longer. Our data show that a two-stranded ␤-sheet (''␤-hairpin'') becomes more stable when the strands are lengthened from five to seven residues, but that further strand lengthening to nine residues does not lead to further ␤-hairpin stabilization for several extension sequences examined. (In one case, all-threonine extension, there may be an additional stabilization on strand lengthening from seven to nine residues.) These results suggest that there may be an intrinsic limit to strand length for most sequences in antiparallel ␤-sheet secondary structure.M ost proteins must fold to a specific three-dimensional shape to perform their biological functions. There is great interest in identifying the factors that determine native conformations; however, despite considerable study, it is not yet possible to predict tertiary folding patterns on the basis of primary structure. A few secondary structures (especially ␣-helix and ␤-sheet) recur throughout known protein structures, and understanding the forces that control conformational preferences within the common secondary structures should contribute to our understanding of conformational preferences at tertiary and quaternary levels. The ␣-helix has been very carefully scrutinized because there are well-established design principles for creating synthetic peptides that adopt ␣-helical secondary structure in the absence of a specific tertiary context (1-7). Until recently, the lack of autonomously folding ␤-sheet model systems made it impossible to conduct analogous studies with this secondary structure (8). In the past several years, however, a number of short peptides (9-24 residues) that display double-or triple-stranded antiparallel ␤-sheet conformations in aqueous solution have been reported (9-11). These model systems provide thermodynamic (12-23) and kinetic (24) insights on ␤-sheet folding behavior. [Solvent-exposed ␤-sheets in specific tertiary contexts have provided a complementary approach for elucidation of ␤-sheet conformational preferences (25,26)]. Here, we show how small designed peptides can be used to assess an aspect of ␤-sheet stability that has not previously been addressed experimentally.␣-Helices become more stable as the length of the helix increases (5-7). This length-dependent effect on conformational stability arises because helix initiation is thermodynamically unfavorable but helix propagation is favorable, at least for some residues (1, 2). Analogous length-dependent stabilization is observed for double-helical nuclei...

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Incorporation of chemical probes into proteins is a powerful way to elucidate biological processes and to engineer novel function. Here we describe an approach that allows ligation of synthetic molecules to target proteins in an intracellular environment. A cellular protein is genetically tagged with one-half of a split intein. The complementary half is linked in vitro to the synthetic probe, and this fusion is delivered into cells using a transduction peptide. Association of the intein halves in the cytosol triggers protein trans-splicing, resulting in the ligation of the probe to the target protein through a peptide bond. This process is specific and applicable to cytosolic and integral membrane proteins. The technology should allow cellular proteins to be elaborated with a variety of abiotic probes.