Christine Berger

ANTIGEN RECOGNITION BY CONFORMATIONAL SELECTION

Conformational adaptation between antigen and antibody can modulate the antibody specificity. The phenomenon has often been proposed to result from an ‘induced fit’, which implies that the binding reaction induces a conformational change in the antigen and the antibody. Thus, an ‘induced fit’ requires initial complex formation followed by a conformational change in the complex. However, an antibody may select those antigen molecules that happen to be in a fitting conformational state. This leads to the same end result as an induced fit. Here, we demonstrate conformational selection by a single chain antibody fragment, raised against a random coil variant of the leucine zipper domain of transcription factor GCN4, when it cross‐reacts with the wild‐type dimeric leucine zipper. Kinetic and equilibrium data show that the single chain antibody fragment fragment selects monomeric peptides from the population in equilibrium with the leucine zipper dimer.

Diffusion-controlled DNA recognition by an unfolded, monomeric bZIP transcription factor

Basic leucine zipper (bZIP) transcription factors are dimers that recognize mainly palindromic DNA sites. It has been assumed that bZIP factors have to form a dimer in order to bind to their target DNA. We find that DNA binding of both monomeric and dimeric bZIP transcription factor GCN4 is diffusion‐limited and that, therefore, the rate of dimerization of the bZIP domain does not affect the rate of DNA recognition and GCN4 need not dimerize in order to bind to its specific DNA site. The results have implications for the mechanism by which bZIP transcription factors find their target sites for transcriptional regulation.

The Amino Acid Sequence of Equine Metallothioneins

The determination of the amino acid sequences of metallothioneins has been undertaken with the aim of obtaining information on 1) the distribution of metal-liganding cysteinyl residues in the polypeptide chain, 2) the mode of metal-protein interaction, 3) the relationship between chemical structure and metal-ion selectivity, 4) the structural basis of chromatographic polymorphism, 5) the identity or difference of metallothioneins from different organs, and 6) the evolution of metallothioneins. The answers to these questions were also expected to provide some further clues for the understanding of the physiological function of metallothionein.

Dynamic structure of metallothionein

Molecular sieve chromatography of rabbit liver metallothionein at different electrolyte concentrations revealed that this protein undergoes an increase in Stokes radius from 1.50 to 1.78 nm when the ionic strength is lowered from 0.5 to 0.015 indicating a change in molecular shape and/or hydration. The variation in ionic strength also affects the far‐UV circular dichroism of metallothionein reflecting a conformational transition in the protein. The effects are attributed to changes in intramolecular repulsion between the strongly negatively charged metal‐thiolate clusters of the protein. It is suggested that metallothionein exists in at least two interchangeable conformational states which differ in hydrodynamic properties and whose equilibrium concentrations are determined by the electrostatic free energy of the system.

Nucleotide binding to creatine kinase: an isothermal titration microcalorimetry study

We investigated the binding of ATP in the presence and absence of Mg2+ to dimeric muscle creatine kinase (CK) by isothermal titration microcalorimetry as a function of pH and temperature. The thermodynamic parameters for these events show that (1) binding of nucleotide to the CK active site does not involve proton exchange with the buffer and (2) the active sites are the only nucleotide binding sites on CK. Interdependence of the active sites in the dimer could not be demonstrated. As CK undergoes major structural changes upon Mg‐nucleotide binding, a thermodynamic cycle was employed to calculate the contributions of domain movements to the observed enthalpies.

The Amino-Terminal Sequence of a Rat Liver Metallothionein (MT-2)

In 1976 Kojima et al. (1) reported the first amino acid sequence of a metallothionein, equine renal MT-1B. Since then, the primary structures have been determined also for the form MT-1A of equine kidney (2,3) as well as for some metallothioneins of the liver of horse (3), man (4,5), rabbit (6), and mouse (7,8). From comparison of the various equine and human sequences we suggested tentatively that cells from euplacental mammals contain two different cistrons coding for metallothionein which may have evolved by duplication from an ancestral gene (5). This view has now received support also from the recently completed sequence studies of the two mouse isometallothioneins (7,8). In this paper, we report the N-terminal amino acid sequence of another rodent isometallothionein, rat MT-2, and document its very close relationship to mouse-MT-II.

Erratum to: Diffusion-controlled DNA recognition by an unfolded, monomeric bZIP transcription factor (FEBS 19923): [FEBS Letters 425 (1998) 14–18]1

Apparent dissociation constant Kapp of complexes of GCN4 derivatives with CRE19 obtained by £uorescence titration. *Sequence di¡erences to wild-type GCN4 are underlined. The C-terminal extensions were introduced to produce stable disul¢de-linked dimers. The alanine substitutions in AAGCN4 destabilize the leucine zipper by about 8 kJ/mol. Ac, NK-acetyl; StBu, thio-tert.-butyl protection group. The large error of Kapp shows that the experimental data are not adequately described by the simple binding model used for data analysis (Section 2) because it does not account for the complex between monomeric peptide and DNA (equilibrium 3 in Fig. 1). Dissociation constant for binding to AP-120. The CRE and AP-1 sites are in bold. NBD indicates a phosphorothioate-linked (7-nitrobenzo-2-oxa-1,3-diazol-4-yl) group.

Erratum to: Diffusion-controlled DNA recognition by an unfolded, monomeric bZIP transcription factor (FEBS 19923): [FEBS Letters 425 (1998) 14–18]1PII S0014-5793(98)00156-21

Apparent dissociation constant Kapp of complexes of GCN4 derivatives with CRE19 obtained by £uorescence titration. *Sequence di¡erences to wild-type GCN4 are underlined. The C-terminal extensions were introduced to produce stable disul¢de-linked dimers. The alanine substitutions in AAGCN4 destabilize the leucine zipper by about 8 kJ/mol. Ac, NK-acetyl; StBu, thio-tert.-butyl protection group. The large error of Kapp shows that the experimental data are not adequately described by the simple binding model used for data analysis (Section 2) because it does not account for the complex between monomeric peptide and DNA (equilibrium 3 in Fig. 1). Dissociation constant for binding to AP-120. The CRE and AP-1 sites are in bold. NBD indicates a phosphorothioate-linked (7-nitrobenzo-2-oxa-1,3-diazol-4-yl) group.

Erratum to: Diffusion‐controlled DNA recognition by an unfolded, monomeric bZIP transcription factor (FEBS 19923)

Apparent dissociation constant Kapp of complexes of GCN4 derivatives with CRE19 obtained by £uorescence titration. *Sequence di¡erences to wild-type GCN4 are underlined. The C-terminal extensions were introduced to produce stable disul¢de-linked dimers. The alanine substitutions in AAGCN4 destabilize the leucine zipper by about 8 kJ/mol. Ac, NK-acetyl; StBu, thio-tert.-butyl protection group. The large error of Kapp shows that the experimental data are not adequately described by the simple binding model used for data analysis (Section 2) because it does not account for the complex between monomeric peptide and DNA (equilibrium 3 in Fig. 1). Dissociation constant for binding to AP-120. The CRE and AP-1 sites are in bold. NBD indicates a phosphorothioate-linked (7-nitrobenzo-2-oxa-1,3-diazol-4-yl) group.