Metal-dependent stabilization of an active HMG protein

Anthony J. Bell, Hong Xin, Susann Taudte, Zhengshuang Shi, Neville R. Kallenbach

Research output: Contribution to journalArticle

Abstract

Using a cloned single domain of the high mobility group protein 1 (HMGB1), we evaluated the effect of introducing metal binding site(s) on protein stability and function. An HMG domain is a conserved sequence of ∼80 amino acids rich in basic, aromatic and proline residues that is active in binding DNA in a sequence- or structure-specific manner. The design strategy focuses on anchoring selected regions of the protein, specifically loops and turns in the molecule, using His-metal ligands. Changes in secondary structure, thermostability and DNA binding properties of a series of such mutants were evaluated. The two most stable mutant constructs contain three surface histidine replacements (two metal binding sites) in the regions encompassing both turns of the molecule. On ligation with the divalent nickel cation, the stability of these two triple histidine mutants (I38H/N51H/D55H and G39H/N51H/D55H) increases by 1.3 and 1.6 kcal/mol, respectively, relative to the wild-type protein, although the creation of binding sites per se destabilizes the protein. The DNA-binding properties of the modified proteins are not impaired by the introduction of the metal binding motifs. These results indicate that it is feasible to stabilize protein tertiary structure using appropriate placement of surface His-metal bonds without loss of function.

Original languageEnglish (US)
Pages (from-to)817-825
Number of pages9
JournalProtein Engineering
Volume15
Issue number10
StatePublished - Oct 1 2003

Fingerprint

High Mobility Group Proteins
Stabilization
Metals
Proteins
Binding sites
Binding Sites
Histidine
DNA
HMGB1 Protein
Conserved Sequence
Protein Stability
Divalent Cations
Nickel
Molecules
Tertiary Protein Structure
Proline
Ligation
Cations
Ligands
Amino Acids

Keywords

  • Circular dichroism
  • Divalent metals
  • DNA binding
  • High mobility group protein box B (rHMGB1b)
  • Protein stability

ASJC Scopus subject areas

  • Molecular Biology
  • Biochemistry

Cite this

Bell, A. J., Xin, H., Taudte, S., Shi, Z., & Kallenbach, N. R. (2003). Metal-dependent stabilization of an active HMG protein. Protein Engineering, 15(10), 817-825.

Metal-dependent stabilization of an active HMG protein. / Bell, Anthony J.; Xin, Hong; Taudte, Susann; Shi, Zhengshuang; Kallenbach, Neville R.

In: Protein Engineering, Vol. 15, No. 10, 01.10.2003, p. 817-825.

Research output: Contribution to journalArticle

Bell, AJ, Xin, H, Taudte, S, Shi, Z & Kallenbach, NR 2003, 'Metal-dependent stabilization of an active HMG protein', Protein Engineering, vol. 15, no. 10, pp. 817-825.
Bell AJ, Xin H, Taudte S, Shi Z, Kallenbach NR. Metal-dependent stabilization of an active HMG protein. Protein Engineering. 2003 Oct 1;15(10):817-825.
Bell, Anthony J. ; Xin, Hong ; Taudte, Susann ; Shi, Zhengshuang ; Kallenbach, Neville R. / Metal-dependent stabilization of an active HMG protein. In: Protein Engineering. 2003 ; Vol. 15, No. 10. pp. 817-825.
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