Metal-dependent stabilization of an active HMG protein

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

Research output: Contribution to journalArticlepeer-review


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
Issue number10
StatePublished - Oct 1 2003


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

ASJC Scopus subject areas

  • Biochemistry
  • Molecular Biology


Dive into the research topics of 'Metal-dependent stabilization of an active HMG protein'. Together they form a unique fingerprint.

Cite this