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 Å 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 °C and possesses near wild-type ribonuclease activity. The 2.25 Å 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 ∼2 μM, cleaving pBn is predicted to increase the fraction of folded molecules and thus enhance ribonuclease activity at 37 °C. This study suggests that introducing conformational strain by permutation, and releasing strain by cleavage, is a potential mechanism for engineering an artificial zymogen.

A Circularly Permuted Photoactive Yellow Protein as a Scaffold for Photoswitch Design

“…Based on the data described here, it is likely that the conformational dynamics of this insert and particularly its accessibility will be affected by the large changes occurring in PYP. Based on previous experience with loop insertion, a wide range of peptide sequences and lengths are likely to be tolerated at this site (37, 44). Of course, inserts with defined conformational preferences may couple to PYP in a manner quite distinct from the short flexible linker studied here.…”

Construction of Allosteric Protein Switches by Alternate Frame Folding and Intermolecular Fragment Exchange

“…2a), which we spanned with a linker consisting of 30 amino acids [4]. If the N-to-C distance is not known precisely, it is best to err on the side of length, as we have found that using linkers longer than necessary does not dramatically destabilize the CP, in contrast to using linkers that are too short [16]. …”

Crystal Structure of Circular Permuted RoCBM21 (CP90): Dimerisation and Proximity of Binding Sites

“…This mutant, whose N- terminus starts with G90 of Ro CBM21, was named CP90 [33]. Unlike most other constructed circularly permutated proteins [36]–[39], CP90 had significantly enhanced binding affinity and improved selectivity toward long-chain carbohydrates (i.e., its affinity for β-cyclodextrin is less than that for amylose EX-I, which is less than that for soluble and insoluble starches) [33]. For the study reported herein, examination of the CP90 crystal structure, its biophysical properties, and its binding affinity for amylose EX-I has suggested a possible, alternative binding path that causes its altered binding behavior.…”