Basic research on protein structure, function, and regulation has produced the following results: (1) Heat-induced reversible partial unfolding of dodecameric Mn-glutamine synthetase from Escherichia coli at pH 7 involves transitions of two major thermodynamic domains, as evidenced by spectral and calorimetric measurements. Active-site ligands stabilize these domains. Interactions between domains occur and the cooperative unit is the dodecamer (rather than each subunit) in the partial unfolding transitions; (delta)H = 185 kcal/(mol dodecamer). (2) Zinc ion interactions with (a) isolated regulatory dimers of E. coli aspartate transcarbamoylase (ATCase) and ATCase, (b) transcriptional factor IIIA (TFIIIA) from immature oocytes of Xenopus laevis, and (c) yeast arginase have been studied by utilizing high-affinity, sensitive metallochromic indicators. Zinc ion binding stabilizes tertiary structures in (a-c) and also oligomer quaternary structures in (a) and (c); in (b) different affinity classes of zinc ion binding sites in the 9 Zn-fingers of TFIIIA were found and these could have a regulatory role in gene recognition. (3) Fluorescence and spectral studies of zinc-dependent structures of TFIIIA are in progress. A specific labeling of Cys 287 of TFIIIA with a fluorescent probe was achieved by reacting 7S particle (in which all zinc clusters and all but one Cys are buried in the TFIIIA,5S RNA complex) with a thiol reactive reagent. TFIIIA interactions with labeled DNA fragments of the 5S RNA gene and purified TFIIIC now can be studied.

Agency
National Institute of Health (NIH)
Institute
National Heart, Lung, and Blood Institute (NHLBI)
Type
Intramural Research (Z01)
Project #
1Z01HL000204-23
Application #
3878872
Study Section
Project Start
Project End
Budget Start
Budget End
Support Year
23
Fiscal Year
1990
Total Cost
Indirect Cost
Name
National Heart, Lung, and Blood Institute
Department
Type
DUNS #
City
State
Country
United States
Zip Code