Structural and Thermodynamic Analysis of a Conformationally Strained Circular Permutant of Barnase
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 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 2 ?M, 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.
- Research Organization:
- Brookhaven National Lab. (BNL), Upton, NY (United States). National Synchrotron Light Source
- Sponsoring Organization:
- Doe - Office Of Science
- DOE Contract Number:
- DE-AC02-98CH10886
- OSTI ID:
- 980499
- Report Number(s):
- BNL-93417-2010-JA; TRN: US201015%%1884
- Journal Information:
- Biochemistry, Vol. 48
- Country of Publication:
- United States
- Language:
- English
Similar Records
Structural and Functional Consequences of Circular Permutation on the Active Site of Old Yellow Enzyme
Structural redesign of lipase B from Candida antarctica by circular permutation and incremental truncation.