ABSTRACT
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 alpha-helix length and alpha-helix stability, which would be impossible to probe with alpha-helices embedded in folded proteins. Here, we use designed peptides to examine the effect of strand length on antiparallel beta-sheet stability. alpha-Helices become more stable as they grow longer. Our data show that a two-stranded beta-sheet ("beta-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 beta-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 beta-sheet secondary structure.
Subject(s)
Peptides/chemistry , Protein Structure, Secondary , Nuclear Magnetic Resonance, Biomolecular/methods , Oligopeptides/chemistryABSTRACT
We describe the physicochemical characterization of various circular and linear forms of the approximately 60 residue N-terminal Src homology 3 (SH3) domain from the murine c-Crk adapter protein. Structural, dynamic, thermodynamic, kinetic and biochemical studies reveal that backbone circularization does not prevent the adoption of the natural folded structure in any of the circular proteins. Both the folding and unfolding rate of the protein increased slightly upon circularization. Circularization did not lead to a significant thermodynamic stabilization of the full-length protein, suggesting that destabilizing enthalpic effects (e.g. strain) negate the expected favorable entropic contribution to overall stability. In contrast, we find circularization results in a dramatic stabilization of a truncated version of the SH3 domain lacking a key glutamate residue. The ability to rescue the destabilized mutant indicates that circularization may be a useful tool in protein engineering programs geared towards generating minimized proteins.
Subject(s)
Protein Engineering , Protein Folding , Proto-Oncogene Proteins/chemistry , src Homology Domains , Amino Acid Sequence , Animals , Circular Dichroism , Cyclization , Glutamic Acid/genetics , Glutamic Acid/metabolism , Kinetics , Ligands , Mice , Models, Molecular , Molecular Sequence Data , Molecular Weight , Protein Denaturation/drug effects , Proto-Oncogene Proteins/genetics , Proto-Oncogene Proteins c-crk , Spectrometry, Fluorescence , Thermodynamics , Urea/pharmacology , src Homology Domains/drug effectsABSTRACT
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.
