Insights into dimerization and four-helix bundle formation found by dissection of the dimer interface of the GrpE protein from Escherichia coli.
The GrpE heat shock protein from Escherichia coli has a homodimeric structure. The dimer interface encompasses two long alpha-helices at the NH(2)-terminal end from each monomer (forming a "tail"), which lead into a small four-helix bundle from which each monomer contributes two short sequential alpha-helices in an antiparallel topological arrangement. We have created a number of different deletion mutants of GrpE that have portions of the dimer interface to investigate requirements for dimerization and to study four-helix bundle formation. Using chemical crosslinking and analytical ultracentrifugation techniques to probe for multimeric states, we find that a mutant containing only the long alpha-helical tail portion (GrpE1-88) is unable to form a dimer, most likely due to a decrease in alpha-helical content as determined by circular dichroism spectroscopy, thus one reason for a dimeric structure for the GrpE protein is to support the tail region. Mutants containing both of the short alpha-helices (GrpE1-138 and GrpE88-197) are able to form a dimer and presumably the four-helix bundle at the dimer interface. These two mutants have equilibrium constants for the monomer-dimer equilibrium that are very similar to the full-length protein suggesting that the tail region does not contribute significantly to the stability of the dimer. Interestingly, one mutant that contains just one of the short alpha-helices (GrpE1-112) exists as a tetrameric species, which presumably is forming a four-helix bundle structure. A proposed model is discussed for this mutant and its relevance for factors influencing four-helix bundle formation.
Mehl
Andrew F
AF
Department of Chemistry, Knox College, Galesburg, Illinois 61401, USA. amehl@knox.edu
Heskett
Luke D
LD
Jain
Sumesh S
SS
Demeler
Borries
B
D001426:Bacterial Proteins / Q000096:biosynthesis / Q000737:chemistry* / Q000235:genetics; D002850:Chromatography, Gel; D002942:Circular Dichroism; D003432:Cross-Linking Reagents; D019281:Dimerization; D004591:Electrophoresis, Polyacrylamide Gel; D029968:Escherichia coli Proteins / Q000737:chemistry* / Q000235:genetics; D006360:Heat-Shock Proteins / Q000096:biosynthesis / Q000737:chemistry* / Q000235:genetics; D013058:Mass Spectrometry; D008958:Models, Molecular; D009010:Monte Carlo Method; D009154:Mutation; D017384:Sequence Deletion; D014461:Ultracentrifugation
D001426:Bacterial Proteins; D003432:Cross-Linking Reagents; D029968:Escherichia coli Proteins; C068113:GrpE protein, Bacteria; C489400:GrpE protein, E coli; D006360:Heat-Shock Proteins