Structural and thermodynamic analysis of a conformationally strained circular permutant of barnase

Abstract

Circular permutation of a protein covalently links its original termini and creates new ends at another location. To maintain the stability of the permuted structure, the termini are typically bridged by a peptide long enough to span the original distance between them. Here, we take the opposite approach and employ a very short linker to introduce conformational strain into a protein by forcing its termini together. We join the N- and C-termini of the small ribonuclease barnase (normally 27.2 A distant) with a single Cys residue and introduce new termini at a surface loop, to create pBn. Compared to a similar variant permuted with an 18-residue linker, permutation with a single amino acid dramatically destabilizes barnase. Surprisingly, pBn is folded at 10 degrees C and possesses near wild-type ribonuclease activity. The 2.25 A 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 proportional to their thermodynamic stabilities, i.e., the fraction of folded molecules. The stability and activity of cleaved pBn are dependent on protein concentration. At concentrations above approximately 2 microM, cleaving pBn is predicted to increase the fraction of folded molecules and thus enhance ribonuclease activity at 37 degrees C. This study suggests that introducing conformational strain by permutation, and releasing strain by cleavage, is a potential mechanism for engineering an artificial zymogen.

Figure 1

Circular permutation-induced strain mechanism. (A) Effect of permuting a hypothetical five-stranded β 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 permuted using a long peptide linker (red line). New termini are generated at the loop connecting strands 3 and 4 (red arrow). The resulting relaxed permutant (structure 2) is stable and active. A peptide linker shorter than the original N-terminal-C-terminal distance is employed in structure 3 to generate the strained permutant. Depending on the extent of strain, the protein may simply be destabilized, folded but distorted such that activity is 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 introduced by permutation, and the C_α-C_α distance between original 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 red, and linker residues boldface black.

Figure 2

Thermodynamic stability of Bn variants. (A) Urea-induced denaturation curves monitored by Trp fluorescence at 10 °C. Lines are best fits of the data to the two-state linear extrapolation equation. (B) Thermal denaturation curves monitored by CD ellipticity at 230 nm. Symbols are the same as in panel A. Data are normalized assuming two-state unfolding. Lines are meant to guide the eye only.

Figure 3

Binding and refolding of the 1-66 and 67-110 fragments of Bn. (A) Temperature dependence of binding, monitored by Trp fluorescence. Fragments were mixed in a 1:1 ratio at the concentrations indicated. Lines are best fits of the data to the 1:1 binding equation. Fitted K_d values are 30 nM (10 °C), 0.13 μM (20 °C), 1.9 μM (30 °C), and 60 μM (37 °C). (B) Binding isotherm of Bn fragments at 37 °C, calculated using a K_d of 60 μM. The dashed line depicts the values for uncleaved pBn.

Figure 4

RNase activity assays of Bn variants at 10 °C. Hydrolysis of Torula yeast genomic RNA is monitored by the change in absorbance at 260 nm. Error bars are standard deviations of three measurements.

Figure 5

Crystal structure of pBn (magenta) bound to barstar (peach). The perspective is the same as in Figure 1B. The inset shows the 2F_o - F_c composite omit map of the linker loop, contoured at 1.5σ. Nascent termini introduced by permutation are designated N’ and C’.

Figure 6

Superposition of pBn (magenta) and WT Bn (green) crystal structures. (A) Main chain atoms are shown in the same perspective as in Figure 1B. Boxes indicate regions of interest discussed in the text. (B) Close-up of the catalytic site, including the general acid His102. (C) Close-up of the surface loop that contains the nascent N’- and C’-termini (Ser67 and Lys66, respectively).

Figure 7

Structure of the linker region, showing the distribution of strain. Ala1, Gln2, and Arg110 are highlighted to illustrate the large shift in the positions of linker residues (Arg110-Cys0-Ala1-Gln2-Val3-Ile4-Asn5-Thr6) upon permutation. By contrast, side chains of residues Phe7 and Leu109 remain anchored in hydrophobic pockets.

Figure 8

Free energy diagram of the permutation strain mechanism for Bn at 37 °C. Arrows are not drawn to scale.