PDBsum entry 1ab7

Ribonuclease inhibitor

PDB id: 1ab7

Contents

Protein chain

89 a.a. *

* Residue conservation analysis

PDB id:

1ab7

Name:

Ribonuclease inhibitor

Title:

Nmr 15n relaxation and structural studies reveal conformational exchange in barstar c40/82a, 30 structures

Structure:

Barstar. Chain: a. Engineered: yes. Mutation: yes

Source:

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

NMR struc:

30 models

Authors:

K.B.Wong,A.R.Fersht,S.M.V.Freund

Key ref:

K.B.Wong et al. (1997). NMR 15N relaxation and structural studies reveal slow conformational exchange in barstar C40/82A. J Mol Biol, 268, 494-511. PubMed id: 9159486 DOI: 10.1006/jmbi.1997.0989

Date:

04-Feb-97 Release date: 04-Sep-97

Protein chain

P11540  (BARS_BACAM) -  Barstar from Bacillus amyloliquefaciens

Seq: 90 a.a.

Struc: 89 a.a.*

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

Enzyme reactions

• Enzyme class: E.C.?

DOI no: 10.1006/jmbi.1997.0989

J Mol Biol 268:494-511 (1997)

PubMed id: 9159486

NMR 15N relaxation and structural studies reveal slow conformational exchange in barstar C40/82A.

K.B.Wong, A.R.Fersht, S.M.Freund.

ABSTRACT

Barstar an 89-residue protein consisting of four helices and a three-stranded parallel beta-sheet, is the intracellular inhibitor of the endoribonuclease barnase. Barstar C40/82A, a mutant in which the two cysteine residues have been replaced by alanine, has been used as a pseudo wild-type in folding studies and in the crystal structure of the barnase:barstar C40/82A complex. We have determined a high resolution solution structure of barstar C40/82A. The structures of barstar C40/82A and the wild-type are superimposable. A comparison with the crystal structure of the barnase:barstar C40/82A complex revealed subtle differences in the regions involved in the binding of barstar to barnase. Side-chain rotations of residues Asn33, Asp35 and Asp39 and a movement of the binding loop (Pro27-Glu32) towards the binding site of barnase facilitate the formation of interface hydrogen bonds and aromatic contacts in the complex. Extreme line broadening and missing signals in 1H-15N correlation spectra indicate substantial conformational exchange for a large subset of residues. 15N relaxation data at two magnetic field strengths, 11.74 T and 14.10 T, were used to estimate exchange contributions and to map the spectral density function at five frequencies: 0, 50, 60, 450 and 540 MHz. Based on these results, model-free calculations with the inclusion of estimated exchange contributions were used to derive order parameters and internal correlation times. The validity of this approach has been investigated with model-free calculations that incorporate longitudinal relaxation rates and heteronuclear 1H-15N NOE data only at 11.74 T and 14.10 T. The relaxation data suggest substantial conformational exchange in regions of barstar C40/82A, including the binding loop, the second and the third helices, and the second and the third strands. Amide proton exchange experiments suggest a stable hydrogen bond network for all helices and sheets except the third helix and the C-terminal of the second and the third strands. The combined results indicate a rigid body movement of the second helix and twisting motions of the beta-sheet of barstar, which might be important for the interaction with barnase.

Selected figure(s)

Figure 10.
Figure 10. Stereoview of the barstar/barnase interface. Grey lines indicate the co-ordinates of the crystal structure of the barnase:barstar C40/82A complex. Black lines show the overlaid restrained minimised mean structure of barstar C40/82A. Only the co-ordinates of barnase are shown in a ball and stick view. The residues are named according to their residue number. B* and Bn denote barstar C40/82A and barnase, respectively. Broken lines indicate hydrogen bonds involved in binding of the two proteins.

Figure 11.
Figure 11. Ribbon representation of barstar C40/82A showing regions with conformational exchange. Residues with chemical exchange contribution are coded red. Residues whose amide signals are not observable under the condition chosen are coded blue. Side-chains of residues (Trp53, Phe56, Leu71, Ile86 and Leu88) involved in the hydrophobic cluster at the C terminus of β-sheet are shown. The C terminus, helices and strands are labelled.

The above figures are reprinted by permission from Elsevier: J Mol Biol (1997, 268, 494-511) copyright 1997.

Figures were selected by an automated process.