PDBsum entry: 1u9r

Hydrolase

PDB id: 1u9r

Contents

Protein chain

129 a.a. *

Waters ×32

* Residue conservation analysis

PDB id:

1u9r

Name:

Hydrolase

Title:

Crystal structure of staphylococcal nuclease mutant v66e/p117g/h124l/s128a at room temperature

Structure:

Thermonuclease. Chain: a. Synonym: tnase, micrococcal nuclease, staphylococcal nuclease. Engineered: yes. Mutation: yes

Source:

Staphylococcus aureus. Organism_taxid: 1280. Gene: nuc. Expressed in: escherichia coli. Expression_system_taxid: 562. Expression_system_cell_line: ar120.

Resolution:

2.10Å R-factor: 0.193 R-free: 0.217

Authors:

V.P.Denisov,J.L.Schlessman,B.E.Garcia-Moreno,B.Halle

Key ref:

V.P.Denisov et al. (2004). Stabilization of internal charges in a protein: water penetration or conformational change? Biophys J, 87, 3982-3994. PubMed id: 15377517 DOI: 10.1529/biophysj.104.048454

Date:

10-Aug-04 Release date: 21-Dec-04

Protein chain

P00644  (NUC_STAAU) -  Thermonuclease

Seq: 231 a.a.

Struc: 129 a.a.*

* PDB and UniProt seqs differ at 4 residue positions (black crosses)

Enzyme reactions

• Enzyme class: E.C.3.1.31.1 - Micrococcal nuclease.
• Reaction: Endonucleolytic cleavage to nucleoside 3'-phosphates and 3'-phosphooligonucleotide end-products.

 Gene Ontology (GO) functional annotation 

• Biochemical function: nucleic acid binding (3 terms)

DOI no: 10.1529/biophysj.104.048454

Biophys J 87:3982-3994 (2004)

PubMed id: 15377517

Stabilization of internal charges in a protein: water penetration or conformational change?

V.P.Denisov, J.L.Schlessman, B.García-Moreno E, B.Halle.

ABSTRACT

The ionizable amino acid side chains of proteins are usually located at the surface. However, in some proteins an ionizable group is embedded in an apolar internal region. Such buried ionizable groups destabilize the protein and may trigger conformational changes in response to pH variations. Because of the prohibitive energetic cost of transferring a charged group from water to an apolar medium, other stabilizing factors must be invoked, such as ionization-induced water penetration or structural changes. To examine the role of water penetration, we have measured the 17O and 2H magnetic relaxation dispersions (MRD) for the V66E and V66K mutants of staphylococcal nuclease, where glutamic acid and lysine residues are buried in predominantly apolar environments. At neutral pH, where these residues are uncharged, we find no evidence of buried water molecules near the mutation site. This contrasts with a previous cryogenic crystal structure of the V66E mutant, but is consistent with the room-temperature crystal structure reported here. MRD measurements at different pH values show that ionization of Glu-66 or Lys-66 is not accompanied by penetration of long-lived water molecules. On the other hand, the MRD data are consistent with a local conformational change in response to ionization of the internal residues.