PDBsum entry 1bnf

Endonuclease

PDB id: 1bnf

Contents

Protein chains

108 a.a. *

Waters ×228

* Residue conservation analysis

PDB id:

1bnf

Name:

Endonuclease

Title:

Barnase t70c/s92c disulfide mutant

Structure:

Barnase. Chain: a, b, c. Engineered: yes

Source:

Bacillus amyloliquefaciens. Organism_taxid: 1390. Gene: barnase. Expressed in: escherichia coli. Expression_system_taxid: 562.

Resolution:

2.00Å R-factor: 0.173

Authors:

J.Clarke,K.Henrick,A.R.Fersht

Key ref:

J.Clarke et al. (1995). Disulfide mutants of barnase. I: Changes in stability and structure assessed by biophysical methods and X-ray crystallography. J Mol Biol, 253, 493-504. PubMed id: 7473729 DOI: 10.1006/jmbi.1995.0568

Date:

31-Mar-95 Release date: 10-Jul-95

Protein chains

P00648  (RNBR_BACAM) -  Ribonuclease from Bacillus amyloliquefaciens

Seq: 157 a.a.

Struc: 108 a.a.*

* PDB and UniProt seqs differ at 2 residue positions (black crosses)

Enzyme reactions

• Enzyme class: E.C.3.1.27.- - ?????

DOI no: 10.1006/jmbi.1995.0568

J Mol Biol 253:493-504 (1995)

PubMed id: 7473729

Disulfide mutants of barnase. I: Changes in stability and structure assessed by biophysical methods and X-ray crystallography.

J.Clarke, K.Henrick, A.R.Fersht.

ABSTRACT

In this series of papers, we examine the effects of introducing disulfide bonds on the properties, structure and thermodynamics of a small globular protein, barnase. Three mutants have been made, in each of which a single crosslink confers different properties. Two of the disulfide bonds, between residues 43 and 80 (43-80) and between residues 85 and 102 (85-102), stabilise the protein, relative to both wild-type and the corresponding (reduced) dithiol forms: 85-102 is more stable than predicted from the entropic destabilisation of the unfolded state; 43-80 is less stable than predicted. The third disulfide bond, between residues 70 and 92 (70-92) destabilises the protein relative to both wild-type and the corresponding dithiol form, implying significant disruption of the folded protein on formation of the disulfide bond. Crystal structures of the three mutant proteins have been solved. All three proteins have essentially the same fold as wild-type, but with left-handed disulfide bonds, which have dihedral geometries that have not been observed in naturally occurring disulfides. In the very stable mutant 85-102, there is no significant difference between the mutant and wild-type structures: these data do not explain the large stability of this protein. The disulfide bond at 43-80 induces small structural rearrangements close to the site of the disulfide bond, associated with some local disorder: the crosslink appears to decrease the stability of the native form of the protein. The destabilising disulfide bond at 70-92 induces considerable structural change, with displacement of a loop and consequent disruption of a stabilising salt-bridge. Our studies do not support the view that the conformation of the disulfide bond is crucial in determining the stability of the mutant proteins.

Selected figure(s)

Figure 1.
Figure 1. General view of wild-type barnase showing the sites of introduction of disulfide bonds between residues 43 and 80 (43--80), 70 and 92 (70--92) and 85 and 102 (85--102). Picture prepared using the program MOLSCRIPT (Kraulis, 1991).

Figure 7.
Figure 7. The site of the disulfide bond of 70--92. A, Superposition of chain C of 70--92 on chain C of wild- type (red). There are two different conformations of mutant observed, type I (yellow) and type II (green) (Table 5). These differ in the conformation of the disulfide bond. B, Superposition of chain C of 70--92, type I only (yellow), on chain C of wild-type (red), some side- chain N atoms are shown in blue. The salt-bridge between Arg 69 and Asp 93 in wild-type is disrupted in the mutant. In the mutant crystal structure the side-chain is solvated and incompletely defined. In the mutant the side-chain of Lys66 occupies the cavity left by Arg69, but no hydrogen bonds are formed with Asp93.

The above figures are reprinted by permission from Elsevier: J Mol Biol (1995, 253, 493-504) copyright 1995.

Figures were selected by an automated process.