SYNTHESIS AND CHARACTERIZATION OF SUPRAMOLECULAR PROTEIN AGGREGATES -SELF-ASSEMBLED, MOLECULARLY-ORDERED, TUBES FROM ELECTROSTATIC COMPLEMENTATION OF GLUTAMINE-SYNTHETASE DODECAMERS

Dodecameric Escherichia coli glutamine synthetase (GS) is formed from identical subunits arranged in face-to-face hexameric rings, In the pr esence of Zn2+ and other transition metal ions the individual dodecame rs 'stack' to form protein tubes, Previous results have suggested that six binuclear intermolecular metal binding sites are generated at eac h dodecamer-dodecamer interface by juxtaposition of the N-terminal hel ices of each subunit adjacent to an analogous helix from a docked dode camer, In principle, replacement of one of the metal binding sites wit hin each pair of helices with charged amino acids could generate elect rostatic interactions that would provide the basis for heterospecific protein-protein interactions, In turn, this would allow for ordered as sembly of protein tubes with alternating, chemically distinguishable, components, This hypothesis was tested by replacement of one of the me tal-ligating histidines (His12) with aspartic acid, arginine or cystei ne, The H12C mutant was further elaborated by selective thiol modifica tion, with either of the charged reagents 2-iodo-acetic acid or 2-chlo ro-acetamidine, which yield glutamate (H12C-IA) or arginine (H12C-CA) mimics at position 12, Light scattering and electron microscopy were u sed to monitor the 'stacking ability' of these variants in the presenc e of Zn2+. No, or few GS 'tubes' were observed in solutions containing only H12D, H12R, H12C-CA or H12C-IA, in the presence or absence of Zn 2+. In contrast, in mixtures containing H12C-CA and either H12D or H12 C-IA, the complementary GS variants stack in the presence of 100 mu M Zn2+, with apparent second order rate constants that are comparable to the wild type dodecamers. Fluorescence energy transfer experiments wi th fluorescein-labeled H12C-IA (donor) and rhodamine-labeled H12C-CA ( acceptor) were performed and compared with the energy transfer efficie ncy with mixtures containing variable ratios of acceptor-labeled and d onor-labeled wild type GS; the wild type mixtures provide a benchmark for the extent of energy transfer expected in random linear arrangemen ts of donor and acceptor, The efficiency of metal-dependent energy tra nsfer in mixtures containing the acceptor-labeled H12C-CA and the dono r-labeled H12C-IA was 3.2-fold greater than expected for a random dist ribution of charged variants, Together, the results indicate that the charged variants provide a mechanism for heterospecific interaction be tween chemically distinguishable dodecamers that align in an ordered o ne-dimensional array.