546 Abstracts

H033 THE 'ZINC-FINGER' DOMAIN OF THE GLUCOCORTI- COlD RECEPTOR - EFFICIENT OVEREXPRESSION AND FLUORIMETRIC STUDIES OF DNA BINDING

Andrea Richter, Simone Pittelkow and Klaus H. R6hm

Institute of Physiological Chemistry, Philipps-University W-3550 Marburg(Lahn), Germany

The DNA-binding domains of nuclear steroid receptors contain two

metal centers, each consisting of a Zn 2+ ion tetrahedrally coordinated to

four cysteine ligands. The domain of the mammalian glucocorticoid

receptor has been studied most extensively. Although structures of the

free domain [1] and of complexes with synthetic DNA fragments [2]

were solved, very little quantitative information is available concerning

specific functions of the metal ion in folding and stabilization of the domain.

To be able to study DNA binding under well-defined conditions, i.e. controlled metal concentration, ionic strength, pH, etc., we developed

a fluorimetric assay that allows direct kinetic studies of DNA binding in

the nanomolar range. The test is based on a fluorigenic dye associated

with a synthetic hormone response element. When the protein binds to the DNA, fluorescence emission of the bound dye is enhanced due to

increased shielding from the solvent. - In addition, we established an

improved expression system for the receptor domain which considerably

facilitates purification and reconstitution of functional protein. Using

site-directed mutagenesis, we introduced a tryptophan residue into the

hydrophobic core of the protein to afford a spectroscopic probe for fol-

ding studies. Results obtained with these systems will be discussed.

1. T. H~rd, E. Kellenbach, E.Boelens, R.A.Maler, K.Dahlman, L.P.Freed-

man, J.Carlstedt-Duke, K.R. Yamamoto, J.A. Gustafsson, and R. Kaptein Science 249, 157 (1990)

2. B.F. Luisi, W.X. Xu, Z. Otwinowski, L.P. Freedman, K.R. Yamamoto, and P.B. Sigler, Nature 352, 497 (1991)