The high-mobility-group protein 1 box domain (HMGI-BD) is a structural element found in several DNA-binding proteins in eukaryotic cells. Its structure is dominated by three a-helices. The spatial arrangement of these helices into an L-shaped molecule is maintained by a number of apolar residues organized into a main and a secondary hydrophobic core. To analyze the significance of these residues for proper folding, conformational stability, and ability to bind and bend DNA, we have mutated the highly conserved Trpl4 of the Chironomus HMGl a protein and have synthesized a series of N-terminally truncated forms. The observed alterations in DNA-binding and DNA-bending characteristics were correlated with structural consequences, as revealed by CD spectroscopy, limited trypsin digestion, and transverse urea gradient gel electrophoresis. Mutation of the Trpl4 residue (Chironomus [W14A]HMGla) and deletion of the seven N-terminal residues, respectively, which are members of the main and the secondary core of Chironomus HMGla, both resulted in a substantial unfolding of the protein. Unexpectedly, these mutants still retained their ability to bind and bend DNA. Conformational analysis of wildtype cHMGla and [W14A]cHMGIa showed that the proteins unfold at 2-4 M urea. In contrast, their DNA complexes persisted even at 6-8 M of the denaturant. Multiple contacts between the HMGl-BD and the DNA are probably responsible for the unusual stability of the complexes.Keywords: high-mobility-group protein 1 (HMGI) ; high-mobility-group-protein-I-box domain; DNA binding; DNA bending; protein folding.The high-mobility-group-protein-1 -box domain (HMGI -BD) protein family comprises more than 100 known proteins interacting with DNA (reviewed in [l]). They can be roughly grouped into two subfamilies (reviewed in [2, 31). The first subfamily represents molecules that recognize specific DNA sequences and occurs usually in particular types of cells or specific stages of development. The abundant nonhistone chromosomal proteins in eukaryotic cells, the high-mobility-group proteins 1 and 2 (HMGI and 2), are the most prominent members of the vecond subfamily. They comprise proteins exhibiting binding preference for A+T-rich, bent, distorted, negatively supercoiled, cruciform DNAs, and do not possess a marked DNA-sequence specificity.The HMGl-BDs are L-shaped molecules 14-61 comprising three helices with angles of about 80 degrees between the antiparallel helices I and IT, and helix 111. Recently, modes of sequence-specific binding of HMG1-BDs of the human sex-deterCorrespondence to J. R. Wiiniewski, 111.

Es wird am Beispiel des 42K+‐Kations in wäßriger KNO3‐Lösung das Verfahren der direkten Messung der elektrischen Beweglichkeit radioaktiver Ionen in Lösungen beschrieben. Die Bedingungen für die Durchführung und die Genauigkeit der Methode werden besprochen und die Meßergebnisse mitgeteilt. Ein wesentliches Kennzeichen ist der Wegfall der Indikatorlösung, die sonst bei der Methode der Grenzflächenwanderung eine wichtige Rolle spielt. Der relative Isotopieeffekt der elektrischen Beweglichkeit, bezogen auf das Isotopengemisch 39K+‐ und 41K+‐Ion (natürliches Verhältnis) zu 42K+‐Ion, beträgt für 0,01 bis 0,1 molare KNO3‐Lösungen bei 25 °C rund 2,30/00. Mit dieser Differenz sind auch die ermittelten Überführungszahlen des K+‐Ions behaftet.