Natl. folded at 10 C and possesses near wild-type ribonuclease activity. The 2 2.25 ? X-ray crystal structure of pBn reveals how the barnase fold is able to adapt to permutation, partially defuse conformational strain, and preserve enzymatic function. We demonstrate that strain in pBn can be relieved by cleaving the linker with a chemical reagent. Catalytic activity of both uncleaved (strained) pBn and cleaved (relaxed) pBn is usually proportional to their thermodynamic stabilities, i.e., the portion of folded molecules. The stability and activity of cleaved pBn are dependent on protein concentration. At concentrations above 2 sheet in Physique 1A. The three-dimensional structure is typically preserved in all other aspects. Indeed, most previous studies attempt to preserve the stability and structure of the permuted protein. The new termini are placed in a solvent-exposed loop, and the original ends are bridged by a peptide long enough to span the distance observed in the wild-type (WT)1 structure. These steps have produced stable and functional permutants of proteins such as GFP and related variants (4, 5), SH3 domains (6), PDZ domains (7), antibody light chain (8), and many others (e.g., refs 9-15). Open in a separate window Physique 1 Circular permutation-induced strain mechanism. (A) Effect of permuting a hypothetical five-stranded sheet using progressively shorter linker peptides. N- and C-termini are designated by a circle and an arrow, respectively. The WT protein (structure 1) is usually permuted using a long peptide linker (reddish collection). New termini are generated at the loop connecting strands 3 and 4 (reddish arrow). The producing relaxed permutant (structure 2) is usually stable and active. A peptide linker shorter than the initial N-terminal-C-terminal Bis-PEG4-acid distance is employed in structure 3 to generate the strained permutant. Depending on the extent of strain, the protein may just be destabilized, folded but distorted such that activity is usually diminished, or unfolded. Cleavage of the short linker relieves strain and allows the protein to refold as a complex (structure 4). (B) X-ray structure of WT Bn (green) complexed with the competitive inhibitor barstar (peach) (44). The locations of new termini Rabbit Polyclonal to SFRS11 launched by permutation, and the C-C distance between initial termini, are indicated. (C) Amino acid sequences of Bn variants used in this study. Amino acids are numbered according to the WT sequence. Residues 1-66 are colored blue, residues 67-110 reddish, and linker residues boldface black. Here, we take the opposite approach and expose strain by deliberately forcing the original termini together. This mechanism is usually shown schematically in Physique 1A. If the WT protein (structure 1) is usually permuted with a long linker, the producing permutant (structure 2) is usually relaxed, relatively stable, and functional. Permutation with a short connecting peptide introduces conformational strain (structure 3). The magnitude of strain is usually expected to depend on the distance discrepancy between the termini of the WT structure and the ends of the new surface loop in the permuted structure. If that disparity is usually small, then strain may destabilize the protein without appreciably perturbing its structure. A larger discrepancy may result in distortion of the active site structure Bis-PEG4-acid such that activity is usually diminished or lost. In the extreme case, the new covalent linkage may compress the original termini to the point where it is no longer possible for the protein to remain folded. Cleaving the linker by chemical or enzymatic means is usually then envisioned to allow the original termini to unwind to their favored positions, permitting the protein to refold as a complex of two fragments (structure 4). We test the Bis-PEG4-acid permutation-strain mechanism using the bacterial ribonuclease barnase (Bn). Bn is usually a suitable target because it is usually small, stable, and known to unfold reversibly under a variety of conditions, including after it has been cleaved into two fragments (16, 17). In addition, its enzymatic activity is usually harmful to both prokaryotic and eukaryotic cells, a feature which makes it of potential therapeutic interest (18, 19). Using Bn as a model system, we inquire two questions. Can permutation-induced strain significantly perturb protein structure? If so, can we reverse the perturbation such that enzyme function can be controlled? To address those issues, it is necessary to understand how the native structure responds to permutation-induced strain. Are structural changes localized to the nascent loop and termini, or are they distributed throughout the protein? How much distortion can be tolerated without global unfolding? How strong is the native fold? We pursue these structural questions by X-ray crystallographic studies of a strained Bn permutant in complex with the natural inhibitor barstar. EXPERIMENTAL PROCEDURES Protein Expression and Purification Bn variants were coexpressed with barstar in BL21(DE3). Following induction with IPTG at 20 C, cells were harvested by centrifugation and lysed with a small amount.