PDBsum entry: 1hz9

Transcription

PDB id: 1hz9

Contents

Protein chains

66 a.a. *

Waters ×126

* Residue conservation analysis

PDB id:

1hz9

Name:

Transcription

Title:

Bacillus caldolyticus cold-shock protein mutants to study determinants of protein stability

Structure:

Cold shock protein cspb. Chain: a, b. Synonym: cspb, bc-csp. Engineered: yes. Mutation: yes

Source:

Bacillus caldolyticus. Organism_taxid: 1394. Gene: cspb. Expressed in: escherichia coli. Expression_system_taxid: 562

Resolution:

1.80Å R-factor: 0.200 R-free: 0.234

Authors:

H.Delbrueck,U.Mueller,D.Perl,F.X.Schmid,U.Heinemann

Key ref:

H.Delbrück et al. (2001). Crystal structures of mutant forms of the Bacillus caldolyticus cold shock protein differing in thermal stability. J Mol Biol, 313, 359-369. PubMed id: 11800562 DOI: 10.1006/jmbi.2001.5051

Date:

24-Jan-01 Release date: 07-Nov-01

Protein chains

P41016  (CSPB_BACCL) -  Cold shock protein CspB

Seq: 66 a.a.

Struc: 66 a.a.*

* PDB and UniProt seqs differ at 1 residue position (black cross)

 Gene Ontology (GO) functional annotation 

• Cellular component: cytoplasm (1 term)
• Biological process: response to stress (4 terms)
• Biochemical function: nucleic acid binding (2 terms)

DOI no: 10.1006/jmbi.2001.5051

J Mol Biol 313:359-369 (2001)

PubMed id: 11800562

Crystal structures of mutant forms of the Bacillus caldolyticus cold shock protein differing in thermal stability.

H.Delbrück, U.Mueller, D.Perl, F.X.Schmid, U.Heinemann.

ABSTRACT

The cold shock proteins Bc-Csp from the thermophile Bacillus caldolyticus and Bs-CspB from the mesophile Bacillus subtilis differ significantly in their conformational stability, although the two proteins differ by only 12 out of 67 amino acid residues. The three-dimensional structure of these small and compact beta-barrel proteins without disulfide bonds, cis-proline residues or tightly bound cofactors is very similar. Previous work has shown that Bc-Csp displays a twofold increase in the free energy of stabilization relative to its homolog Bs-CspB, and indicated that electrostatic interactions are, in part, responsible for this effect. It was further described that the stability difference is almost exclusively due to surface-exposed charged residues at sequence positions 3 and 66 of Bc-Csp and Bs-CspB, whereas all other amino acid changes between both proteins have no net effect on stability. To investigate how two surface residues determine the stability of Bc-Csp, Arg3 and Leu66 were replaced by glutamic acid, corresponding to the Bs-CspB sequence. The crystal structures of the resultant protein variants, Bc-Csp R3E and Bc-Csp L66E, were determined at 1.4 A and 1.27 A resolution, and refined to R values of 13.9 % and 15.8 %, respectively. Both structures closely resemble Bc-Csp in their global fold and show different hydrogen bonding and salt-bridge patterns when two independent molecules in the asymmetric unit of the crystal are compared. To extend the study to neighbored residues that help determine the surface charge around Arg3 and Leu66, the mutant proteins Bc-Csp E46A, Bc-Csp R3E/E46A/L66E and Bc-Csp V64T/L66E/67A were crystallized. Their structures were determined at resolutions of 1.8 A, 1.32 A and 1.8 A and refined to R values of 18.5 %, 13.8 % and 19.3 %, respectively. A systematic comparison of the crystal structures of all forms of the B. caldolyticus cold shock protein shows varying patterns of hydrogen bonds and electrostatic interactions around residues 3 and 66. Thermal destabilization of the protein by mutation appears to correlate with the extent of an acidic surface patch near the C-terminal carboxylate group.

Selected figure(s)

Figure 1.
Figure 1. Three-dimensional structure and sequence of the B. caldolyticus cold shock protein, Bc-Csp. The drawing shows the structure of Bc-Csp in two orientations (top). The residues mutated in this study cluster in one area of the protein surface and are labeled. In the right panel, the b-strands b1 to b5 and the connecting loops L1 to L4 are labeled as well. The amino acid sequence of Bc-Csp is compared with the sequence of the cold shock protein from B. subtilis, Bs-CspB (bottom). Sequence differences are highlighted in color. Mutations in residues marked red have little influence on protein stability[10]. Residues marked green are mutated in the crystal structures reported here. The residues mutated in Bc-Csp R3E/E46A/L66E or in Bc-Csp V64T/L66E/67A are printed in italics or underlined, respectively.

Figure 4.
Figure 4. Distribution of surface charges in Bc-Csp and its mutated variants. The molecules are oriented with the surface area bearing the mutations facing forward. A drawing of Bc-Csp is shown in the same orientation for comparison.

The above figures are reprinted by permission from Elsevier: J Mol Biol (2001, 313, 359-369) copyright 2001.

Figures were selected by the author.