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 presence of Zn2+ and other transition metal ions the individual dodecamers 'stack' to form protein tubes. Previous results have suggested that six binuclear intermolecular metal binding sites are generated at each dodecamer-dodecamer interface by juxtaposition of the N-terminal helices of each subunit adjacent to an analogous helix from a docked dodecamer. In principle, replacement of one of the metal binding sites within each pair of helices with charged amino acids could generate electrostatic interactions that would provide the basis for heterospecific protein-protein interactions. In turn, this would allow for ordered assembly of protein tubes with alternating, chemically distinguishable, components. This hypothesis was tested by replacement of one of the metalligating histidines (His12) with aspartic acid, arginine or cysteine. The H12C mutant was further elaborated by selective thiol modification, with either of the charged reagents 2-iodo-acetic acid or 2-chloro-acetamidine, which yield glutamate (H12C-IA) or arginine (H12C-CA) mimics at position 12. Light scattering and electron microscopy were used to monitor the 'stacking ability' of these variants in the presence 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 Zn2+. In contrast, in mixtures containing H12C-CA and either H12D or H12C-IA, the complementary GS variants stack in the presence of 100 microM Zn2+, with apparent second order rate constants that are comparable to the wild type dodecamers. Fluorescence energy transfer experiments with fluorescein-labeled H12C-IA (donor) and rhodamine-labeled H12C-CA (acceptor) were performed and compared with the energy transfer efficiency with mixtures containing variable ratios of acceptor-labeled and donor-labeled wild type GS; the wild type mixtures provide a benchmark for the extent of energy transfer expected in random linear arrangements of donor and acceptor. The efficiency of metal-dependent energy transfer in mixtures containing the acceptor-labeled H12C-CA and the donor-labeled H12C-IA was 3.2-fold greater than expected for a random distribution of charged variants. Together, the results indicate that the charged variants provide a mechanism for heterospecific interaction between chemically distinguishable dodecamers that align in an ordered one-dimensional array.
Chen
J P
JP
The Department of Medicinal Chemistry, University of Washington, Seattle 98195-7610, USA.
Dabrowski
M J
MJ
Atkins
W M
WM
D001665:Binding Sites; D004563:Electrochemistry; D004926:Escherichia coli / Q000201:enzymology*; D005974:Glutamate-Ammonia Ligase / Q000737:chemistry* / Q000378:metabolism; D006639:Histidine / Q000737:chemistry; D007700:Kinetics; D008027:Light; D046911:Macromolecular Substances; D008854:Microscopy, Electron; D016296:Mutagenesis; D017433:Protein Structure, Secondary; D012542:Scattering, Radiation; D013050:Spectrometry, Fluorescence; D013329:Structure-Activity Relationship; D015032:Zinc / Q000494:pharmacology
D046911:Macromolecular Substances; D006639:Histidine; D005974:Glutamate-Ammonia Ligase; D015032:Zinc