Molecular interactions in paracrystals of a fragment corresponding to the alpha-helical coiled-coil rod portion of glial fibrillary acidic protein: evidence for an antiparallel packing of molecules and polymorphism related to intermediate filament structure.
Open Access
- 1 July 1989
- journal article
- research article
- Published by Rockefeller University Press in The Journal of cell biology
- Vol. 109 (1), 225-234
- https://doi.org/10.1083/jcb.109.1.225
Abstract
We have expressed in Escherichia coli a fragment of c-DNA that broadly corresponds to the alpha-helical coiled-coil rod section of glial fibrillary acidic protein (GFAP) and have used the resultant protein to prepare paracrystals in which molecular interactions can be investigated. An engineered fragment of mouse GFAP c-DNA was inserted into a modified version of the E. coli expression vector pLcII, from which large quantities of a lambda cII-GFAP rod fusion protein were prepared. A protein fragment corresponding to the GFAP rod was then obtained by proteolysis with thrombin. Paracrystals of this material were produced using divalent cations (Mg, Ca, Ba) in the presence of a chaotrophic agent such as thiocyanate. These paracrystals showed a number of polymorphic patterns that were based on a fundamental pattern that had dyad symmetry and an axial repeat of 57 nm. Analysis of both positive and negative staining patterns showed that this fundamental pattern was consistent with a unit cell containing two 48-nm-long molecules in an antiparallel arrangement with their NH2 termini overlapping by approximately 34 nm. More complicated patterns were produced by stacking the fundamental pattern with staggers of approximately 1/5, 2/5, and 1/2 the axial repeat. The molecular packing the unit cell was consistent with a range of solution studies on intermediate filaments that have indicated that a molecular dimer (i.e., a tetramer containing four chains or two coiled-coil molecules) is an intermediate in filament assembly. Moreover, these paracrystals allow the molecular interactions involved in the tetramer to be investigated in some detail.This publication has 58 references indexed in Scilit:
- Expression in Escherichia coli of fragments of glial fibrillary acidic protein: Characterization, assembly properties and paracrystal formationJournal of Cell Science, 1989
- Two distinct attachment sites for vimentin along the plasma membrane and the nuclear envelope in avian erythrocytes: a basis for a vectorial assembly of intermediate filaments.The Journal of cell biology, 1987
- Intermediate Filaments: Conformity and Diversity of Expression and StructureAnnual Review of Cell Biology, 1985
- Antiparallel orientation of the two double-stranded coiled-coils in the tetrameric protofilament unit of intermediate filamentsJournal of Molecular Biology, 1985
- A periodic ultrastructure in intermediate filamentsJournal of Molecular Biology, 1982
- DISASSEMBLY OF SYNTHETIC 10-NM DESMIN FILAMENTS FROM SMOOTH-MUSCLE INTO PROTOFILAMENTS1981
- Tropomyosin coiled-coil interactions: Evidence for an unstaggered structureJournal of Molecular Biology, 1975
- Cleavage of Structural Proteins during the Assembly of the Head of Bacteriophage T4Nature, 1970
- PERIODIC REPEAT UNITS OF EPITHELIAL CELL TONOFILAMENTSThe Journal of cell biology, 1967
- Tropomyosin Paracrystals Formed by Divalent CationsScience, 1966