STRUCTURAL AND FUNCTIONAL CONSEQUENCES OF MUTATIONS WITHIN THE HYDROPHOBIC CORES OF THE HMG1-BOX DOMAIN OF THE CHIRONOMUS HIGH-MOBILITY-GROUP PROTEIN 1A
Jr. Wisniewski et al., STRUCTURAL AND FUNCTIONAL CONSEQUENCES OF MUTATIONS WITHIN THE HYDROPHOBIC CORES OF THE HMG1-BOX DOMAIN OF THE CHIRONOMUS HIGH-MOBILITY-GROUP PROTEIN 1A, European journal of biochemistry, 243(1-2), 1997, pp. 151-159
The high-mobility-group protein 1 box domain (HMG1-BD) is a structural
element found in several DNA-binding proteins in eukaryotic cells. It
s structure is dominated by three a-helices. The spatial arrangement o
f these helices into an L-shaped molecule is maintained by a number of
apolar residues organized into a main and a secondary hydrophobic cor
e. To analyze the significance of these residues for proper folding, c
onformational stability, and ability to bind and bend DNA, we have mut
ated the highly conserved Trp14 of the Chironomus HMG1a protein and ha
ve synthesized a series of N-terminally truncated forms. The observed
alterations in DNA-binding and DNA-bending characteristics were correl
ated with structural consequences, as revealed by CD spectroscopy, lim
ited trypsin digestion, and transverse urea gradient gel electrophores
is. Mutation of the Trp14 residue (Chironomus [W14A]HMG1a) and deletio
n of the seven N-terminal residues, respectively, which are members of
the main and the secondary core of Chironomus HMG1a, both resulted in
a substantial unfolding of the protein. Unexpectedly, these mutants s
till retained their ability to bind and bend DNA. Conformational analy
sis of wildtype cHMG1a and [W14A]cHMG1a showed that the proteins unfol
d at 2-4 M urea. In contrast, their DNA complexes persisted even at 6-
8 M of the denaturant. Multiple contacts between the HMG1-BD and the D
NA are probably responsible for the unusual stability of the complexes
.